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Background: The immunological determinants of delayed viral clearance and intrahost viral evolution that drive the development of new pathogenic virus strains in immunocompromised individuals are unknown. Therefore, we longitudinally studied severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)-specific immune responses in relation to viral clearance and evolution in immunocompromised individuals., Methods: Among Omicron-infected immunocompromised individuals, we determined SARS-CoV-2-specific T- and B-cell responses, anti-spike immunoglobulin G (IgG) and IgG3 titers, neutralization titers, and monoclonal antibody (mAb) resistance-associated mutations. The 28-day post-enrollment nasopharyngeal specimen defined early (reverse-transcription polymerase chain reaction [RT-PCR] negative ≤28 days) or late (RT-PCR positive >28 days) viral clearance., Results: Of 30 patients included (median age, 61.9 [interquartile range, 47.4-72.3] years; 50% females), 20 (66.7%) received mAb therapy. Thirteen (43.3%) demonstrated early and 17 (56.7%) late viral clearance. Patients with early viral clearance and patients without resistance-associated mutations had significantly higher baseline interferon-γ release, and patients with early viral clearance had a higher frequency of SARS-CoV-2-specific B cells at baseline. In non-mAb-treated patients, day 7 IgG and neutralization titers were significantly higher in those with early versus late viral clearance., Conclusions: An early robust adaptive immune response is vital for efficient viral clearance and associated with less emergence of mAb resistance-associated mutations in Omicron-infected immunocompromised patients. This emphasizes the importance of early SARS-CoV-2-specific T- and B-cell responses and thereby provides a rationale for development of novel therapeutic approaches., Competing Interests: Potential conflicts of interest. W. J. W. reported receiving grant support from the NWO, grant from the Amsterdam UMC Graduate School outside the submitted work, and personal fees (paid to his institution) from AstraZeneca and Shionogi. E. B. reported receiving grant support from the NWO, outside the submitted work. H. P.-S. reported receiving a Kolff postdoc grant, outside the submitted work. All other authors report no potential 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., (© The Author(s) 2024. Published by Oxford University Press on behalf of Infectious Diseases Society of America.)

Background: The increasing burden of dengue virus on public health due to more explosive and frequent outbreaks highlights the need for improved surveillance and control. Genomic surveillance of dengue virus not only provides important insights into the emergence and spread of genetically diverse serotypes and genotypes, but it is also critical to monitor the effectiveness of newly implemented control strategies. Here, we present DengueSeq, an amplicon sequencing protocol, which enables whole-genome sequencing of all four dengue virus serotypes., Results: We developed primer schemes for the four dengue virus serotypes, which can be combined into a pan-serotype approach. We validated both approaches using genetically diverse virus stocks and clinical specimens that contained a range of virus copies. High genome coverage (>95%) was achieved for all genotypes, except DENV2 (genotype VI) and DENV 4 (genotype IV) sylvatics, with similar performance of the serotype-specific and pan-serotype approaches. The limit of detection to reach 70% coverage was 10-100 RNA copies/μL for all four serotypes, which is similar to other commonly used primer schemes. DengueSeq facilitates the sequencing of samples without known serotypes, allows the detection of multiple serotypes in the same sample, and can be used with a variety of library prep kits and sequencing instruments., Conclusions: DengueSeq was systematically evaluated with virus stocks and clinical specimens spanning the genetic diversity within each of the four dengue virus serotypes. The primer schemes can be plugged into existing amplicon sequencing workflows to facilitate the global need for expanded dengue virus genomic surveillance., (© 2024. The Author(s).)

Mpox is an emerging zoonotic disease which has now spread to over 113 countries as of August 2023, with over 89,500 confirmed human cases. The Netherlands had one of the highest incidence rates in Europe during the peak of the outbreak. In this study, we generated 158 near-complete mpox virus (MPXV) genomes (12.4% of nationwide cases) that were collected throughout the Netherlands from the start of the outbreak in May 2022 to August 2023 to track viral evolution and investigate outbreak dynamics. We detected 14 different viral lineages, suggesting multiple introductions followed by rapid initial spread within the country. The estimated evolutionary rate was relatively high compared to previously described in orthopoxvirus literature, with an estimated 11.58 mutations per year. Genomic rearrangement events occurred at a rate of 0.63% and featured a large deletion event. In addition, based on phylogenetics, we identified multiple potential transmission clusters which could be supported by direct source- and contact tracing data. This led to the identification of at least two main transmission locations at the beginning of the outbreak. We conclude that whole genome sequencing of MPXV is essential to enhance our understanding of outbreak dynamics and evolution of a relatively understudied and emerging zoonotic pathogen., (© 2024 The Authors. Journal of Medical Virology published by Wiley Periodicals LLC.)

Rapid identification of the rise and spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern remains critical for monitoring of the efficacy of diagnostics, therapeutics, vaccines, and control strategies. A wide range of SARS-CoV-2 next-generation sequencing (NGS) methods have been developed over the last years, but cross-sequence technology benchmarking studies have been scarce. In the current study, 26 clinical samples were sequenced using five protocols: AmpliSeq SARS-CoV-2 (Illumina), EasySeq RC-PCR SARS-CoV-2 (Illumina/NimaGen), Ion AmpliSeq SARS-CoV-2 (Thermo Fisher), custom primer sets (Oxford Nanopore Technologies (ONT)), and capture probe-based viral metagenomics (Roche/Illumina). Studied parameters included genome coverage, depth of coverage, amplicon distribution, and variant calling. The median SARS-CoV-2 genome coverage of samples with cycle threshold (Ct) values of 30 and lower ranged from 81.6 to 99.8% for, respectively, the ONT protocol and Illumina AmpliSeq protocol. Correlation of coverage with PCR Ct values varied per protocol. Amplicon distribution signatures differed across the methods, with peak differences of up to 4 log 10 at disbalanced positions in samples with high viral loads (Ct values ≤ 23). Phylogenetic analyses of consensus sequences showed clustering independent of the workflow used. The proportion of SARS-CoV-2 reads in relation to background sequences, as a (cost-)efficiency metric, was the highest for the EasySeq protocol. The hands-on time was the lowest when using EasySeq and ONT protocols, with the latter additionally having the shortest sequence runtime. In conclusion, the studied protocols differed on a variety of the studied metrics. This study provides data that assist laboratories when selecting protocols for their specific setting., (© 2023. The Author(s).)

Objective: Viral infections during pregnancy are a major health concern to mother and fetus. By repurposing cell-free Non Invasive Prenatal Testing (NIPT) sequencing data, we investigated prevalence and abundance of viral DNA in a cohort of 108,349 pregnant women., Method: Cell-free DNA (cfDNA) sequencing reads that did not map to any of the human chromosomes or mitochondrial DNA of the human reference genome build GRCh38 were aligned to 224 DNA viruses selected from the NCBI refseq viral database., Results: In total 443,665 reads of viral origin were detected across 42,273 samples representing 165 viral species. Several are known to be potentially harmful during pregnancy and/or childbirth, including Cytomegalovirus, Parvovirus B19 and Hepatitis B. Viral sequences were mostly detected at very low abundance. However, several cases had exceptionally high viral loads for Parvovirus B19, Hepatitis B and others. We found statistically significant associations between presence of viral DNA and gestational age, maternal age, fetal fraction, cfDNA concentration and others., Conclusion: We demonstrate the feasibility to detect viral DNA from typical genome-wide NIPT cfDNA sequencing and describe the main characteristics of the viral DNA in our cohort. Our dataset of detected viral sequence reads is made publicly available to guide future clinical implementations., (© 2022 The Authors. Prenatal Diagnosis published by John Wiley & Sons Ltd.)

Infections by DNA viruses during pregnancy are associated with increased health risks to both mother and fetus. Although not all DNA viruses are related to an increased risk of complications during pregnancy, several can directly infect the fetus and/or cause placental dysfunction. During Non-Invasive Prenatal Testing analysis, the presence of viral DNA can be detected, theoretically allowing screening early in pregnancy. Although treatment options are currently limited, this might rapidly change in the near future. It is therefore important to be aware of the potential impact of these viruses on feto-maternal health. In this manuscript we provide a brief introduction into the most commonly detected DNA viruses in human cell-free DNA sequencing experiments and their pathogenic potential during pregnancy., (© 2022 The Authors. Prenatal Diagnosis published by John Wiley & Sons Ltd.)

Competing Interests: Declaration of interests We declare no competing interests.

Background: The aim of this randomized, controlled trial is to determine whether antisevere acute respiratory syndrome coronavirus 2 hyperimmune globulin (COVIG) protects against severe coronavirus disease 2019 (COVID-19) in severely immunocompromised, hospitalized, COVID-19 patients., Methods: Patients were randomly assigned to receive COVIG or intravenous immunoglobulin (IVIG) without SARS-CoV-2 antibodies., Results: Severe COVID-19 was observed in 2 of 10 (20%) patients treated with COVIG compared to 7 of 8 (88%) in the IVIG control group (P = .015, Fisher's exact test)., Conclusions: Antisevere acute respiratory syndrome coronavirus 2 hyperimmune globulin may be a valuable treatment in severely immunocompromised, hospitalized, COVID-19 patients and should be considered when no monoclonal antibody therapies are available., Competing Interests: Potential conflicts of interest. F. S. and V. M. J. N. work at Sanquin Blood Supply Foundation. 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., (© The Author(s) 2022. Published by Oxford University Press on behalf of Infectious Diseases Society of America.)

Herpes simplex virus (HSV) and cytomegalovirus (CMV) are DNA viruses that are common among humans. Severely immunocompromised patients are at increased risk of developing HSV or CMV disease due to a weakened immune system. Antiviral therapy can be challenging because these drugs have a narrow therapeutic window and show significant pharmacokinetic variability. Above that, immunocompromised patients have various comorbidities like impaired renal function and are exposed to polypharmacy. This scoping review discusses the current pharmacokinetic (PK) and pharmacodynamic (PD) knowledge of antiviral drugs for HSV and CMV treatment in immunocompromised patients. HSV and CMV treatment guidelines are discussed, and multiple treatment interventions are proposed: early detection of drug resistance; optimization of dose to target concentration by therapeutic drug monitoring (TDM) of nucleoside analogs; the introduction of new antiviral drugs; alternation between compounds with different toxicity profiles; and combinations of synergistic antiviral drugs. This research will also serve as guidance for future research, which should focus on prospective evaluation of the benefit of each of these interventions in randomized controlled trials.

Background: In fall 2020 when schools in the Netherlands operated under a limited set of COVID-19 measures, we conducted outbreaks studies in four secondary schools to gain insight in the level of school transmission and the role of SARS-CoV-2 transmission via air and surfaces., Methods: Outbreak studies were performed between 11 November and 15 December 2020 when the wild-type variant of SARS-CoV-2 was dominant. Clusters of SARS-CoV-2 infections within schools were identified through a prospective school surveillance study. All school contacts of cluster cases, irrespective of symptoms, were invited for PCR testing twice within 48 h and 4-7 days later. Combined NTS and saliva samples were collected at each time point along with data on recent exposure and symptoms. Surface and active air samples were collected in the school environment. All samples were PCR-tested and sequenced when possible., Results: Out of 263 sampled school contacts, 24 tested SARS-CoV-2 positive (secondary attack rate 9.1%), of which 62% remained asymptomatic and 42% had a weakly positive test result. Phylogenetic analysis on 12 subjects from 2 schools indicated a cluster of 8 and 2 secondary cases, respectively, but also other distinct strains within outbreaks. Of 51 collected air and 53 surface samples, none were SARS-CoV-2 positive., Conclusion: Our study confirmed within school SARS-CoV-2 transmission and substantial silent circulation, but also multiple introductions in some cases. Absence of air or surface contamination suggests environmental contamination is not widespread during school outbreaks., (© 2022. The Author(s).)

Monkeypox (MPX) is a disease caused by the monkeypox virus. It is a viral zoonotic disease, endemic in Central and West Africa. Human-to-human spread also occurs and is a feature of the current global outbreak. As far as we know, exponential transmission during this outbreak is not related to changed viral characteristics but due to multiple high-risk contacts in a subset of people that have contracted the virus, so far almost exclusively affecting men who have sex with men (MSM). Appropriate public health measures and increased alertness of all health care providers is needed to increase case-finding and decrease transmission. There is a real chance of MPX to become endemic in large parts of the world.

We report a severe acute respiratory syndrome coronavirus 2 superspreading event in the Netherlands after distancing rules were lifted in nightclubs, despite requiring a negative test or vaccination. This occurrence illustrates the potential for rapid dissemination of variants in largely unvaccinated populations under such conditions. We detected subsequent community transmission of this strain.

We describe the lessons learned during a SARS-CoV-2 variant-of-concern Alpha outbreak investigation at a normal care unit in a university hospital in Amsterdam in December 2020. The outbreak consisted of nine nurses and two roomed-in patient family members. (attack rate 18%). One nurse tested positive with a phylogenetically distinct variant, after a documented infection 83 days prior. Three key points were taken from this investigation. First, it was controlled by adherence to existing guidelines, despite increased transmissibility of the variant. Second, viral sequencing can inform transmission cluster inference, but the epidemiological context is essential to draw appropriate conclusions. Third, reinfections with Alpha variants can occur rapidly after primary infection., (© 2021. The Author(s).)

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections on mink farms are increasingly observed in several countries, leading to the massive culling of animals on affected farms. Recent studies showed multiple (anthropo)zoonotic transmission events between humans and mink on these farms. Mink-derived SARS-CoV-2 sequences from The Netherlands and Denmark contain multiple substitutions in the S protein receptor binding domain (RBD). Molecular modeling showed that these substitutions increase the mean binding energy, suggestive of potential adaptation of the SARS-CoV-2 S protein to the mink angiotensin-converting enzyme 2 (ACE2) receptor. These substitutions could possibly also impact human ACE2 binding affinity as well as humoral immune responses directed to the RBD region of the SARS-CoV-2 S protein in humans. We wish to highlight these observations to raise awareness and urge for the continued surveillance of mink (and other animal)-related infections., (© The Author(s) 2021. Published by Oxford University Press.)

The continuing pandemic threat posed by avian influenza A/H5N1 viruses calls for improved insights into their evolution during human infection. We performed whole genome deep sequencing of respiratory specimens from 44 H5N1-infected individuals from Indonesia and found substantial within-host viral diversity. At nearly 30% of genome positions multiple amino acids were observed within or across samples, including positions implicated in aerosol transmission between ferrets. Amino acid variants detected our cohort were often found more frequently in available H5N1 sequences of human than avian isolates. We additionally identified previously unreported amino acid variants and multiple variants that increased in proportion over time in available sequential samples. Given the importance of the polymerase complex for host adaptation, we tested 121 amino acid variants found in the PB2, PB1 and PA subunits for their effects on polymerase activity in human cells. We identified multiple single amino acid variants in all three polymerase subunits that substantially increase polymerase activity including some with effects comparable to that of the widely recognized adaption and virulence marker PB2-E627 K. These results indicate highly dynamic evolutionary processes during human H5N1 virus infection and the potential existence of previously undocumented adaptive pathways.

Background: We aimed to identify HBc amino acid differences between subgroups of chronic hepatitis B (CHB) patients., Methods: Deep sequencing of HBc was performed in samples of 89 CHB patients (42 HBeAg positive, 47 HBeAg negative). Amino acid types were compared using Sequence Harmony to identify subgroup specific sites between HBeAg-positive and -negative patients, and between patients with combined response and non-response to peginterferon/adefovir combination therapy., Results: We identified 54 positions in HBc where the frequency of appearing amino acids was significantly different between HBeAg-positive and -negative patients. In HBeAg negative patients, 22 positions in HBc were identified which differed between patients with treatment response and those with non-response. The fraction non-consensus sequence on selected positions was significantly higher in HBeAg-negative patients, and was negatively correlated with HBV DNA and HBsAg levels., Conclusions: Sequence Harmony identified a number of amino acid changes associated with HBeAg-status and response to peginterferon/adefovir combination therapy., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Background and Aim: Precore (PC) and basal core promoter (BCP) mutations are associated with responses to interferon-based treatment in HBeAg-positive chronic hepatitis B (CHB) patients. Here, we identify viral minority variants in these regions and assess association with response to peginterferon-alfa (Peg-IFN) and adefovir combination therapy., Patients and Methods: Ultra-deep pyrosequencing analysis of the BCP and PC region was performed for 89 CHB patients (42 HBeAg-positive; 47 HBeAg-negative), at baseline and during treatment. Specifically, associations of individual positions with the HBeAg-negative phenotype were studied, as well as the association of the most prevalent mutations with combined response in HBeAg-positive and -negative patients at week 72 (HBeAg negativity, HBV-DNA <2000 IU/mL and ALT normalization at 24 weeks of treatment-free follow-up)., Results: The mutations most strongly correlated with the HBeAg-negative phenotype were at positions 1762/1764 and 1896/1899 in the BCP and PC region, respectively. No major changes in nucleotide composition of these positions were observed during treatment. In HBeAg-negative patients, a combined presence of 1764A and 1896A was correlated with lower ALT levels (p = 0.004), whereas the presence of 1899A was correlated with higher age (p = 0.030), lower HBV-DNA level (p = 0.036), and previous IFN therapy (p = 0.032). The presence of 1764A/1896A or the absence of 1899A at baseline, was associated with lower response rates, after adjustment for HBV genotype (p = 0.031 and p = 0.017) and HBsAg level (p = 0.035 and p = 0.022)., Conclusion: We identified novel correlations between common BCP and PC variants with response to Peg-IFN and adefovir in HBeAg-negative patients. Ultimately, this may guide the selection of those patients most likely to benefit from Peg-IFN-based treatment., (Copyright © 2017 Elsevier B.V. All rights reserved.)

We compared 454 amplicon sequencing with clonal sequencing for the characterization of intra-host hepatitis C virus (HCV) NS3 variants. Clonal and 454 sequences were obtained from 12 patients enrolled in a clinical phase I study for telaprevir, an NS3-4a protease inhibitor. Thirty-nine datasets were used to compare the consensus sequence, average pairwise distance, normalized Shannon entropy, phylogenetic tree topology and the number and frequency of variants derived from both sequencing techniques. In general, a good concordance was observed between both techniques for the majority of datasets. Discordant results were observed for 5 out of 39 clonal and 454 datasets, which could be attributed to primer-related selective amplification used for clonal sequencing. Both 454 and clonal datasets consisted of a few major variants and a large number of low-frequency variants. Telaprevir resistance-associated variants were observed in low frequencies and were detected more often by 454. We conclude that performance of 454 and clonal sequencing is comparable for the characterization of intra-host virus populations. Not surprisingly, 454 is superior for the detection of low frequency resistance-associated variants. However, despite the greater coverage, 454 failed to detect some low frequency variants detected by clonal sequencing., (Copyright © 2015 Elsevier B.V. All rights reserved.)