Integration of Sequencing and Epidemiologic Data for Surveillance of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Infections in a Tertiary-Care Hospital.

BACKGROUND: The ongoing coronavirus disease 2019 pandemic significantly burdens hospitals and other healthcare facilities. Therefore, understanding the entry and transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is critical for effective prevention and preparedness measures. We performed surveillance and analysis of testing and transmission of SARS-CoV-2 infections in a tertiary-care hospital in Germany during the second and third pandemic waves in fall/winter 2020. METHODS: Between calendar week 41 in 2020 and calendar week 1 in 2021, 40%, of all positive patient and staff samples (284 total) were subjected to full-length viral genome sequencing. Clusters were defined based on similar genotypes indicating common sources of infection. We integrated phylogenetic, spatial, and temporal metadata to detect nosocomial infections and outbreaks, uncover transmission chains, and evaluate containment measures' effectiveness. RESULTS: Epidemiologic data and contact tracing readily recognize most healthcare-associated (HA) patient infections. However, sequencing data reveal that temporally preceding index cases and transmission routes can be missed using epidemiologic methods, resulting in delayed interventions and serially linked outbreaks being counted as independent events. While hospital-associated transmissions were significantly elevated at a moderate rate of community transmission during the second wave, systematic testing and high vaccination rates among staff have led to a substantial decrease in HA infections at the end of the second/beginning of the third wave despite high community transmissions. CONCLUSIONS: While epidemiologic analysis is critical for immediate containment of HA SARS-CoV-2 outbreaks, integration of genomic surveillance revealed weaknesses in identifying staff contacts. Our study underscores the importance of high testing frequency and genomic surveillance to detect, contain and prevent SARS-CoV-2-associated infections in healthcare settings.

Experimental and pan-cancer genome analyses reveal widespread contribution of acrylamide exposure to carcinogenesis in humans.

Humans are frequently exposed to acrylamide, a probable human carcinogen found in commonplace sources such as most heated starchy foods or tobacco smoke. Prior evidence has shown that acrylamide causes cancer in rodents, yet epidemiological studies conducted to date are limited and, thus far, have yielded inconclusive data on association of human cancers with acrylamide exposure. In this study, we experimentally identify a novel and unique mutational signature imprinted by acrylamide through the effects of its reactive metabolite glycidamide. We next show that the glycidamide mutational signature is found in a full one-third of approximately 1600 tumor genomes corresponding to 19 human tumor types from 14 organs. The highest enrichment of the glycidamide signature was observed in the cancers of the lung (88% of the interrogated tumors), liver (73%), kidney (>70%), bile duct (57%), cervix (50%), and, to a lesser extent, additional cancer types. Overall, our study reveals an unexpectedly extensive contribution of acrylamide-associated mutagenesis to human cancers.

Human papillomavirus type 38 alters wild-type p53 activity to promote cell proliferation via the downregulation of integrin alpha 1 expression.

Tumor suppressors can exert pro-proliferation functions in specific contexts. In the beta human papillomavirus type 38 (HPV38) experimental model, the viral proteins E6 and E7 promote accumulation of a wild-type (WT) p53 form in human keratinocytes (HKs), promoting cellular proliferation. Inactivation of p53 by different means strongly decreases the proliferation of HPV38 E6/E7 HKs. This p53 form is phosphorylated at S392 by the double-stranded RNA-dependent protein kinase PKR, which is highly activated by HPV38. PKR-mediated S392 p53 phosphorylation promotes the formation of a p53/DNMT1 complex, which inhibits expression of integrin alpha 1 (ITGA1), a repressor of epidermal growth factor receptor (EGFR) signaling. Ectopic expression of ITGA1 in HPV38 E6/E7 HKs promotes EGFR degradation, inhibition of cellular proliferation, and cellular death. Itga1 expression was also inhibited in the skin of HPV38 transgenic mice that have an elevated susceptibility to UV-induced skin carcinogenesis. In summary, these findings reveal the existence of a specific WT p53 form that displays pro-proliferation properties.

Merkel Cell Polyomavirus Downregulates N-myc Downstream-Regulated Gene 1, Leading to Cellular Proliferation and Migration.

Merkel cell polyomavirus (MCPyV) is the first human polyomavirus etiologically associated with Merkel cell carcinoma (MCC), a rare and aggressive form of skin cancer. Similar to other polyomaviruses, MCPyV encodes early T antigen genes, viral oncogenes required for MCC tumor growth. To identify the unique oncogenic properties of MCPyV, we analyzed the gene expression profiles in human spontaneously immortalized keratinocytes (NIKs) expressing the early genes from six distinct human polyomaviruses (PyVs), including MCPyV. A comparison of the gene expression profiles revealed 28 genes specifically deregulated by MCPyV. In particular, the MCPyV early gene downregulated the expression of the tumor suppressor gene N-myc downstream-regulated gene 1 (NDRG1) in MCPyV gene-expressing NIKs and hTERT-MCPyV gene-expressing human keratinocytes (HK) compared to their expression in the controls. In MCPyV-positive MCC cells, the expression of NDRG1 was downregulated by the MCPyV early gene, as T antigen knockdown rescued the level of NDRG1. In addition, NDRG1 overexpression in hTERT-MCPyV gene-expressing HK or MCC cells resulted in a decrease in the number of cells in S phase and cell proliferation inhibition. Moreover, a decrease in wound healing capacity in hTERT-MCPyV gene-expressing HK was observed. Further analysis revealed that NDRG1 exerts its biological effect in Merkel cell lines by regulating the expression of the cyclin-dependent kinase 2 (CDK2) and cyclin D1 proteins. Overall, NDRG1 plays an important role in MCPyV-induced cellular proliferation.IMPORTANCE Merkel cell carcinoma was first described in 1972 as a neuroendocrine tumor of skin, most cases of which were reported in 2008 to be caused by a PyV named Merkel cell polyomavirus (MCPyV), the first PyV linked to human cancer. Thereafter, numerous studies have been conducted to understand the etiology of this virus-induced carcinogenesis. However, it is still a new field, and much work is needed to understand the molecular pathogenesis of MCC. In the current work, we sought to identify the host genes specifically deregulated by MCPyV, as opposed to other PyVs, in order to better understand the relevance of the genes analyzed on the biological impact and progression of the disease. These findings open newer avenues for targeted drug therapies, thereby providing hope for the management of patients suffering from this highly aggressive cancer.

PVAmpliconFinder: a workflow for the identification of human papillomaviruses from high-throughput amplicon sequencing.

BACKGROUND: The detection of known human papillomaviruses (PVs) from targeted wet-lab approaches has traditionally used PCR-based methods coupled with Sanger sequencing. With the introduction of next-generation sequencing (NGS), these approaches can be revisited to integrate the sequencing power of NGS. Although computational tools have been developed for metagenomic approaches to search for known or novel viruses in NGS data, no appropriate tool is available for the classification and identification of novel viral sequences from data produced by amplicon-based methods. RESULTS: We have developed PVAmpliconFinder, a data analysis workflow designed to rapidly identify and classify known and potentially new Papillomaviridae sequences from NGS amplicon sequencing with degenerate PV primers. Here, we describe the features of PVAmpliconFinder and its implementation using biological data obtained from amplicon sequencing of human skin swab specimens and oral rinses from healthy individuals. CONCLUSIONS: PVAmpliconFinder identified putative new HPV sequences, including one that was validated by wet-lab experiments. PVAmpliconFinder can be easily modified and applied to other viral families. PVAmpliconFinder addresses a gap by providing a solution for the analysis of NGS amplicon sequencing, increasingly used in clinical research. The PVAmpliconFinder workflow, along with its source code, is freely available on the GitHub platform: https://github.com/IARCbioinfo/PVAmpliconFinder.

Detection of a large spectrum of viral infections in conjunctival premalignant and malignant lesions.

To study the interaction between HIV and other carcinogenic infections in conjunctival squamous cell carcinoma (SCC), we evaluated the presence of a broad spectrum of human viruses in conjunctiva specimens. Beta Human papillomavirus (HPV; n = 46), gamma HPV (n = 52), polyomaviruses (n = 12) and herpes viruses (n = 3) was determined in DNA extracted from 67 neoplastic and 55 non-neoplastic conjunctival tissues of HIV-positive and HIV negative subjects by Luminex-based assays. Next-generation sequencing (NGS) was also used to further characterize the presence of cutaneous HPVs. Detection of beta-2 HPV infections was associated with the risk of neoplasia (adjusted odds ratio [aOR] 3.0; 95% confidence interval [CI] 1.3-6.8), regardless of HIV status (HIV positive, aOR 2.6, 95% CI 0.9-7.7; HIV negative, aOR 3.5, 95% CI 0.9-14.4). EBV was strongly associated with the risk of neoplasia (aOR 12.0, 95% CI 4.3-33.5; P < .01) mainly in HIV individuals (HIV positive, aOR 57.5; 95% CI: 10.1-327.1; HIV negative aOR 2.6; 95% CI: 0.2-34.7). NGS allowed to identify 13 putative novel HPVs in cases and controls. Our findings suggest a role of beta HPV types and EBV, in conjunctival SCC. However, additional studies of viral expression in tumor tissue are required to confirm the causal association.

Interplay between the Epigenetic Enzyme Lysine (K)-Specific Demethylase 2B and Epstein-Barr Virus Infection.

The histone modifier lysine (K)-specific demethylase 2B (KDM2B) plays a role in the differentiation of hematopoietic cells, and its expression appears to be deregulated in certain cancers of hematological and lymphoid origins. We have previously found that the KDM2B gene is differentially methylated in cell lines derived from Epstein-Barr virus (EBV)-associated endemic Burkitt lymphoma (eBL) compared with that in EBV-negative sporadic Burkitt lymphoma-derived cells. However, whether KDM2B plays a role in eBL development has not been previously investigated. Oncogenic viruses have been shown to hijack the host cell epigenome to complete their life cycle and to promote the transformation process by perturbing cell chromatin organization. Here, we investigated whether EBV alters KDM2B levels to enable its life cycle and promote B-cell transformation. We show that infection of B cells with EBV leads to downregulation of KDM2B levels. We also show that LMP1, one of the main EBV transforming proteins, induces increased DNMT1 recruitment to the KDM2B gene and augments its methylation. By altering KDM2B levels and performing chromatin immunoprecipitation in EBV-infected B cells, we show that KDM2B is recruited to the EBV gene promoters and inhibits their expression. Furthermore, forced KDM2B expression in immortalized B cells led to altered mRNA levels of some differentiation-related genes. Our data show that EBV deregulates KDM2B levels through an epigenetic mechanism and provide evidence for a role of KDM2B in regulating virus and host cell gene expression, warranting further investigations to assess the role of KDM2B in the process of EBV-mediated lymphomagenesis.IMPORTANCE In Africa, Epstein-Barr virus infection is associated with endemic Burkitt lymphoma, a pediatric cancer. The molecular events leading to its development are poorly understood compared with those leading to sporadic Burkitt lymphoma. In a previous study, by analyzing the DNA methylation changes in endemic compared with sporadic Burkitt lymphoma cell lines, we identified several differential methylated genomic positions in the proximity of genes with a potential role in cancer, and among them was the KDM2B gene. KDM2B encodes a histone H3 demethylase already shown to be involved in some hematological disorders. However, whether KDM2B plays a role in the development of Epstein-Barr virus-mediated lymphoma has not been investigated before. In this study, we show that Epstein-Barr virus deregulates KDM2B expression and describe the underlying mechanisms. We also reveal a role of the demethylase in controlling viral and B-cell gene expression, thus highlighting a novel interaction between the virus and the cellular epigenome.

Beta HPV38 oncoproteins act with a hit-and-run mechanism in ultraviolet radiation-induced skin carcinogenesis in mice.

Cutaneous beta human papillomavirus (HPV) types are suspected to be involved, together with ultraviolet (UV) radiation, in the development of non-melanoma skin cancer (NMSC). Studies in in vitro and in vivo experimental models have highlighted the transforming properties of beta HPV E6 and E7 oncoproteins. However, epidemiological findings indicate that beta HPV types may be required only at an initial stage of carcinogenesis, and may become dispensable after full establishment of NMSC. Here, we further investigate the potential role of beta HPVs in NMSC using a Cre-loxP-based transgenic (Tg) mouse model that expresses beta HPV38 E6 and E7 oncogenes in the basal layer of the skin epidermis and is highly susceptible to UV-induced carcinogenesis. Using whole-exome sequencing, we show that, in contrast to WT animals, when exposed to chronic UV irradiation K14 HPV38 E6/E7 Tg mice accumulate a large number of UV-induced DNA mutations, which increase proportionally with the severity of the skin lesions. The mutation pattern detected in the Tg skin lesions closely resembles that detected in human NMSC, with the highest mutation rate in p53 and Notch genes. Using the Cre-lox recombination system, we observed that deletion of the viral oncogenes after development of UV-induced skin lesions did not affect the tumour growth. Together, these findings support the concept that beta HPV types act only at an initial stage of carcinogenesis, by potentiating the deleterious effects of UV radiation.

Human papillomavirus type 16 antagonizes IRF6 regulation of IL-1ß.

Human papillomavirus type 16 (HPV16) and other oncoviruses have been shown to block innate immune responses and to persist in the host. However, to avoid viral persistence, the immune response attempts to clear the infection. IL-1ß is a powerful cytokine produced when viral motifs are sensed by innate receptors that are members of the inflammasome family. Whether oncoviruses such as HPV16 can activate the inflammasome pathway remains unknown. Here, we show that infection of human keratinocytes with HPV16 induced the secretion of IL-1ß. Yet, upon expression of the viral early genes, IL-1ß transcription was blocked. We went on to show that expression of the viral oncoprotein E6 in human keratinocytes inhibited IRF6 transcription which we revealed regulated IL-1ß promoter activity. Preventing E6 expression using siRNA, or using E6 mutants that prevented degradation of p53, showed that p53 regulated IRF6 transcription. HPV16 abrogation of p53 binding to the IRF6 promoter was shown by ChIP in tissues from patients with cervical cancer. Thus E6 inhibition of IRF6 is an escape strategy used by HPV16 to block the production IL-1ß. Our findings reveal a struggle between oncoviral persistence and host immunity; which is centered on IL-1ß regulation.

Multicenter benchmarking of short and long read wet lab protocols for clinical viral metagenomics.

Metagenomics is gradually being implemented for diagnosing infectious diseases. However, in-depth protocol comparisons for viral detection have been limited to individual sets of experimental workflows and laboratories. In this study, we present a benchmark of metagenomics protocols used in clinical diagnostic laboratories initiated by the European Society for Clinical Virology (ESCV) Network on NGS (ENNGS). A mock viral reference panel was designed to mimic low biomass clinical specimens. The panel was used to assess the performance of twelve metagenomic wet lab protocols currently in use in the diagnostic laboratories of participating ENNGS member institutions. Both Illumina and Nanopore, shotgun and targeted capture probe protocols were included. Performance metrics sensitivity, specificity, and quantitative potential were assessed using a central bioinformatics pipeline. Overall, viral pathogens with loads down to 104 copies/ml (corresponding to CT values of 31 in our PCR assays) were detected by all the evaluated metagenomic wet lab protocols. In contrast, lower abundant mixed viruses of CT values of 35 and higher were detected only by a minority of the protocols. Considering the reference panel as the gold standard, optimal thresholds to define a positive result were determined per protocol, based on the horizontal genome coverage. Implementing these thresholds, sensitivity and specificity of the protocols ranged from 67 to 100 % and 87 to 100 %, respectively. A variety of metagenomic protocols are currently in use in clinical diagnostic laboratories. Detection of low abundant viral pathogens and mixed infections remains a challenge, implying the need for standardization of metagenomic analysis for use in clinical settings.

The E2F4/p130 Repressor Complex Cooperates with Oncogenic ΔNp73α To Inhibit Gene Expression in Human Papillomavirus 38 E6/E7-Transformed Keratinocytes and in Cancer Cells.

Tumor suppressor p53 and its related proteins, p63 and p73, can be synthesized as multiple isoforms lacking part of the N- or C-terminal regions. Specifically, high expression of the ΔNp73α isoform is notoriously associated with various human malignancies characterized by poor prognosis. This isoform is also accumulated by oncogenic viruses, such as Epstein-Barr virus (EBV), as well as genus beta human papillomaviruses (HPV) that appear to be involved in carcinogenesis. To gain additional insight into ΔNp73α mechanisms, we have performed proteomics analyses using human keratinocytes transformed by the E6 and E7 proteins of the beta-HPV type 38 virus as an experimental model (38HK). We find that ΔNp73α associates with the E2F4/p130 repressor complex through a direct interaction with E2F4. This interaction is favored by the N-terminal truncation of p73 characteristic of ΔNp73 isoforms. Moreover, it is independent of the C-terminal splicing status, suggesting that it could represent a general feature of ΔNp73 isoforms (α, ß, γ, δ, ε, ζ, θ, η, and η1). We show that the ΔNp73α-E2F4/p130 complex inhibits the expression of specific genes, including genes encoding for negative regulators of proliferation, both in 38HK and in HPV-negative cancer-derived cell lines. Such genes are not inhibited by E2F4/p130 in primary keratinocytes lacking ΔNp73α, indicating that the interaction with ΔNp73α rewires the E2F4 transcriptional program. In conclusion, we have identified and characterized a novel transcriptional regulatory complex with potential implications in oncogenesis. IMPORTANCE The TP53 gene is mutated in about 50% of human cancers. In contrast, the TP63 and TP73 genes are rarely mutated but rather expressed as ΔNp63 and ΔNp73 isoforms in a wide range of malignancies, where they act as p53 antagonists. Accumulation of ΔNp63 and ΔNp73, which is associated with chemoresistance, can result from infection by oncogenic viruses such as EBV or HPV. Our study focuses on the highly carcinogenic ΔNp73α isoform and uses a viral model of cellular transformation. We unveil a physical interaction between ΔNp73α and the E2F4/p130 complex involved in cell cycle control, which rewires the E2F4/p130 transcriptional program. Our work shows that ΔNp73 isoforms can establish interactions with proteins that do not bind to the TAp73α tumor suppressor. This situation is analogous to the gain-of-function interactions of p53 mutants supporting cellular proliferation.

HPV38 impairs UV-induced transcriptional activation of the IL-18 pro-inflammatory cytokine.

IMPORTANCE: Here, we demonstrate that the direct binding of p53 on the IL-18 promoter region regulates its gene expression. However, the presence of E6 and E7 from human papillomavirus type 38 impairs this mechanism via a new inhibitory complex formed by DNA methyltransferase 1 (DNMT1)/PKR/ΔNp73α, which binds to the region formerly occupied by p53 in primary keratinocytes.

Evaluation of human papillomavirus DNA in colorectal cancer and adjacent mucosal tissue samples.

BACKGROUND: Although the role of viral agents, such as human papillomavirus (e.g. HPV16, HPV18) in colorectal cancer (CRC) has been previously investigated, results remain inconclusive. METHODS: To further evaluate the involvement of oncogenic HPV types in CRC, 40 frozen neoplastic and 40 adjacent colonic tissues collected from Italian patients were analyzed by Luminex-based assays that detect a broad spectrum of HPV types, i.e. Alpha (n = 21), Beta (n = 46) and Gamma HPVs (n = 52). In addition, 125 frozen CRC samples and 70 surrounding mucosal tissues were collected from Czech patients and analyzed by broad spectrum PCR protocols: (i) FAP59/64, (ii) FAPM1 and (iii) CUT combined with Next Generation Sequencing (NGS). RESULTS: Using Luminex-basedassays, DNA from HPV16 was detected in 5% (2/40) CRC tissues from Italian patients. One HPV16 DNA-positive CRC case was subsequently confirmed positive for E6*I mRNA. Cutaneous beta HPV types were detected in 10% (4/40) adjacent tissues only, namely HPV111 (n = 3) and HPV120 (n = 1), while gamma HPV168 (n = 1) and HPV199 (n = 1) types were detected in adjacent and in tumor tissues, respectively. The NGS analysis of the CRC Czech samples identified HPV sequences from mucosal alpha-3 (HPV89), alpha-7 (HPV18, 39, 68 and 70) and alpha-10 species (HPV11), as well as cutaneous beta-1 (HPV20, 24, 93, 98, 105,124) beta-2 (HPV23), beta-3 (HPV49) and gamma-1 species (HPV205). CONCLUSIONS: Our findings indicate that HPV types belonging to the mucosal alpha, and the 'cutaneous' beta and gamma genera can be detected in the colonic mucosal samples with a low prevalence rate and a low number of HPV reads by Luminex and NGS, respectively. However, additional studies are required to corroborate these findings.

Clinical Evaluation of a Fully-Automated High-Throughput Multiplex Screening-Assay to Detect and Differentiate the SARS-CoV-2 B.1.1.529 (Omicron) and B.1.617.2 (Delta) Lineage Variants.

BACKGROUND: The recently emerged SARS-CoV-2 B.1.1.529 lineage and its sublineages (Omicron variant) pose a new challenge to healthcare systems worldwide due to its ability to efficiently spread in immunized populations and its resistance to currently available monoclonal antibody therapies. RT-PCR-based variant tests can be used to screen large sample-sets rapidly and accurately for relevant variants of concern (VOC). The aim of this study was to establish and validate a multiplex assay on the cobas 6800/8800 systems to allow discrimination between the two currently circulating VOCs, Omicron and Delta, in clinical samples. METHODS: Primers and probes were evaluated for multiplex compatibility. Analytic performance was assessed using cell culture supernatant of an Omicron variant isolate and a clinical Delta variant sample, normalized to WHO-Standard. Clinical performance of the multiplex assay was benchmarked against NGS results. RESULTS: In silico testing of all oligos showed no interactions with a high risk of primer-dimer formation or amplification of human DNA/RNA. Over 99.9% of all currently available Omicron variant sequences are a perfect match for at least one of the three Omicron targets included in the multiplex. Analytic sensitivity was determined as 19.0 IU/mL (CI95%: 12.9-132.2 IU/mL) for the A67V + del-HV69-70 target, 193.9 IU/mL (CI95%: 144.7-334.7 IU/mL) for the E484A target, 35.5 IU/mL (CI95%: 23.3-158.0 IU/mL) for the N679K + P681H target and 105.0 IU/mL (CI95%: 80.7-129.3 IU/mL) for the P681R target. All sequence variances were correctly detected in the clinical sample set (225/225 Targets). CONCLUSION: RT-PCR-based variant screening compared to whole genome sequencing is both rapid and reliable in detecting relevant sequence variations in SARS-CoV-2 positive samples to exclude or verify relevant VOCs. This allows short-term decision-making, e.g., for patient treatment or public health measures.

Remdesivir-induced emergence of SARS-CoV2 variants in patients with prolonged infection.

We here investigate the impact of antiviral treatments such as remdesivir on intra-host genomic diversity and emergence of SARS-CoV2 variants in patients with a prolonged course of infection. Sequencing and variant analysis performed in 112 longitudinal respiratory samples from 14 SARS-CoV2-infected patients with severe disease progression show that major frequency variants do not generally arise during prolonged infection. However, remdesivir treatment can increase intra-host genomic diversity and result in the emergence of novel major variant species harboring fixed mutations. This is particularly evident in a patient with B cell depletion who rapidly developed mutations in the RNA-dependent RNA polymerase gene following remdesivir treatment. Remdesivir treatment-associated emergence of novel variants is of great interest in light of current treatment guidelines for hospitalized patients suffering from severe SARS-CoV2 disease, as well as the potential use of remdesivir to preventively treat non-hospitalized patients at high risk for severe disease progression.

Comparing susceptibility and contagiousness in concurrent outbreaks with a non-VOC and the VOC SARS-CoV-2 variant B.1.1.7 in daycare centers in Hamburg, Germany.

We describe two outbreaks of SARS-CoV-2 in daycare centers in the metropolitan area of Hamburg, Germany. The outbreaks occurred in rapid chronological succession, in neighborhoods with a very similar sociodemographic structure, thus allowing for cross-comparison of these events. We combined classical and molecular epidemiologic investigation methods to study infection entry, spread within the facilities, and subsequent transmission of infections to households. Epidemiologic and molecular evidence suggests a superspreading event with a non-variant of concern (non-VOC) SARS CoV-2 strain at the root of the first outbreak. The second outbreak involved two childcare facilities experiencing infection activity with the variant of concern (VOC) B.1.1.7 (Alpha). We show that the index cases in all outbreaks had been childcare workers, and that children contributed substantially to secondary transmission of SARS-CoV-2 infection from childcare facilities to households. The frequency of secondary transmissions in households originating from B.1.1.7-infected children was increased compared to children with non-VOC infections. Self-reported symptoms, particularly cough and rhinitis, occurred more frequently in B.1.1.7-infected children. Especially in light of the rapidly spreading VOC B.1.617.2 (Delta), our data underline the notion that rigorous SARS-CoV-2 testing in combination with screening of contacts regardless of symptoms is an important measure to prevent SARS-CoV-2 infection of unvaccinated individuals in daycare centers and associated households.

MinION nanopore sequencing and assembly of a complete human papillomavirus genome.

BACKGROUND: The MinION sequencer belongs to the third generation of sequencing technology that allows for the generation of ultra-long reads, representing a potentially more effective approach to characterize entire viral genome sequences than other time-consuming and low-throughput methodologies. METHODS: We report the use of the MinION nanopore sequencer to sequence the full-length genome of human papillomavirus (HPV)-ICB2 (7441 bp), which was previously characterized in our laboratory. Three independent MinION libraries were prepared and sequenced using either three consecutive 12 -h runs (Protocol A) or a single run of 48 h starting from a pool of three barcoded DNA libraries (Protocol B). A fully automated bioinformatics pipeline was developed for the reconstruction of the viral genome. RESULTS: Protocols A and B generated 9,354,933 and 3,255,879 reads, respectively. Read length N50 values ranged between 6976 and 7360 nucleotides over the four sequencing runs. Bioinformatics analysis showed that both protocols allowed for the reconstruction of the whole viral genome, with pairwise percentages of identity to HPV-ICB2 of 100 % for protocol A and 99.98 % for protocol B. CONCLUSION: Our results show that the use of the MinION nanopore sequencer represents an effective strategy for whole-genome sequencing of HPVs with a minimal error rate.

Rapid Automated Screening for SARS-CoV-2 B.1.617 Lineage Variants (Delta/Kappa) through a Versatile Toolset of qPCR-Based SNP Detection.

BACKGROUND: The recent emergence of distinct and highly successful SARS-CoV-2 lineages has substantial implications for individual patients and public health measures. While next-generation-sequencing is routinely performed for surveillance purposes, RT-qPCR can be used to rapidly rule-in or rule-out relevant variants, e.g., in outbreak scenarios. The objective of this study was to create an adaptable and comprehensive toolset for multiplexed Spike-gene SNP detection, which was applied to screen for SARS-CoV-2 B.1.617 lineage variants. METHODS: We created a broad set of single nucleotide polymorphism (SNP)-assays including del-Y144/145, E484K, E484Q, P681H, P681R, L452R, and V1176F based on a highly specific multi-LNA (locked nucleic acid)-probe design to maximize mismatch discrimination. As proof-of-concept, a multiplex-test was compiled and validated (SCOV2-617VOC-UCT) including SNP-detection for L452R, P681R, E484K, and E484Q to provide rapid screening capabilities for the novel B.1.617 lineages. RESULTS: For the multiplex-test (SCOV2-617VOC-UCT), the analytic lower limit of detection was determined as 182 IU/mL for L452R, 144 IU/mL for P681R, and 79 IU/mL for E484Q. A total of 233 clinical samples were tested with the assay, including various on-target and off-target sequences. All SNPs (179/179 positive) were correctly identified as determined by SARS-CoV-2 whole genome sequencing. CONCLUSION: The recurrence of SNP locations and flexibility of methodology presented in this study allows for rapid adaptation to current and future variants. Furthermore, the ability to multiplex various SNP-assays into screening panels improves speed and efficiency for variant testing. We show 100% concordance with whole genome sequencing for a B.1.617.2 screening assay on the cobas6800 high-throughput system.

Diversity of human papillomavirus in the anal canal of HIV-positive and HIV-negative men.

BACKGROUND: To characterize the HPV diversity in the anal mucosa of men with different sexual behavior and HIV status by next-generation sequencing (NGS). METHODS: Anal swabs from HIV-positive (n = 94; mean age, 38 years) and HIV-negative (n = 100; mean age, 37.5 years) men who have sex with men (MSM) and HIV-negative men (predominantly men who have sex with women, MSW) (n = 99; mean age, 38.2 years) were analyzed by broad-spectrum PCR protocols combined with NGS. FINDINGS: Alpha HPV types (n = 74) were detected mainly in the MSM groups (HPV6, 11, and 43 were the most abundant types) compared with MSW (n = 16) (HPV11, 32, and 87 were among the most abundant). In contrast, beta HPVs were more abundantly detected among MSW (n = 45) than in the HIV-positive (n = 16) and HIV-negative (n = 26) MSM groups. Gamma HPVs were detected almost equally in HIV-positive MSM (n = 62), HIV-negative MSM (n = 58), and MSW (n = 57). In addition, 31 putative novel PV types were identified. CONCLUSIONS: Our data show that beta and gamma HPV types are present in the anal mucosa, thus reinforcing the existing evidence that they can be detected at anatomical sites other than skin. Alpha and beta HPV distribution among these three groups appears to vary according to sexual behavior.

SARS-CoV-2 Reinfection in a Healthcare Worker Despite the Presence of Detectable Neutralizing Antibodies.

So far, only a few reports about reinfections with SARS-CoV-2 have been published, and they often lack detailed immunological and virological data. We report about a SARS-CoV-2 reinfection with a genetically distinct SARS-CoV-2 variant in an immunocompetent female healthcare worker that has led to a mild disease course. No obvious viral escape mutations were observed in the second virus variant. The infectious virus was shed from the patient during the second infection episode despite the presence of neutralizing antibodies in her blood. Our data indicate that a moderate immune response after the first infection, but not a viral escape, did allow for reinfection and live virus shedding.