Tales from the crypt and coral reef: the successes and challenges of identifying new herpesviruses using metagenomics.

Herpesviruses are ubiquitous double-stranded DNA viruses infecting many animals, with the capacity to cause disease in both immunocompetent and immunocompromised hosts. Different herpesviruses have different cell tropisms, and have been detected in a diverse range of tissues and sample types. Metagenomics-encompassing viromics-analyses the nucleic acid of a tissue or other sample in an unbiased manner, making few or no prior assumptions about which viruses may be present in a sample. This approach has successfully discovered a number of novel herpesviruses. Furthermore, metagenomic analysis can identify herpesviruses with high degrees of sequence divergence from known herpesviruses and does not rely upon culturing large quantities of viral material. Metagenomics has had success in two areas of herpesvirus sequencing: firstly, the discovery of novel exogenous and endogenous herpesviruses in primates, bats and cnidarians; and secondly, in characterizing large areas of the genomes of herpesviruses previously only known from small fragments, revealing unexpected diversity. This review will discuss the successes and challenges of using metagenomics to identify novel herpesviruses, and future directions within the field

Reply to Jensen and Kowalik: Consideration of mixed infections is central to understanding HCMV intrahost diversity.

Kowalik and Jensen (1) have reported that intra-host variation in HCMV approaches levels similar to those of HCV, with fast mutation rates mooted as one explanation (2).While we discussed that HCMV mutation rates were postulated as an explanation for high diversity, the focus of our work is on observed inconsistencies in nucleotide diversity between and within patients (3). Kowalik and Jensen did calculate HCMV mutation rates to be similar to MCMV but maintained that this could underestimate the true levels (2). In contrast, our study showed that in the absence of mixed infections, HCMV is no more diverse than other DNA viruses, and considerably less so than chronic RNA viruses. This simple conclusion is different to that of Kowalik and Jensen and had not been stated prior to our publication.N

Detection of Low Frequency Multi-Drug Resistance and Novel Putative Maribavir Resistance in Immunocompromised Pediatric Patients with Cytomegalovirus.

Human cytomegalovirus (HCMV) is a significant pathogen in immunocompromised individuals, with the potential to cause fatal pneumonitis and colitis, as well as increasing the risk of organ rejection in transplant patients. With the advent of new anti-HCMV drugs there is therefore considerable interest in using virus sequence data to monitor emerging resistance to antiviral drugs in HCMV viraemia and disease, including the identification of putative new mutations. We used target-enrichment to deep sequence HCMV DNA from 11 immunosuppressed pediatric patients receiving single or combination anti-HCMV treatment, serially sampled over 1-27 weeks. Changes in consensus sequence and resistance mutations were analyzed for three ORFs targeted by anti-HCMV drugs and the frequencies of drug resistance mutations monitored. Targeted-enriched sequencing of clinical material detected mutations occurring at frequencies of 2%. Seven patients showed no evidence of drug resistance mutations. Four patients developed drug resistance mutations a mean of 16 weeks after starting treatment. In two patients, multiple resistance mutations accumulated at frequencies of 20% or less, including putative maribavir and ganciclovir resistance mutations P522Q (UL54) and C480F (UL97). In one patient, resistance was detected 14 days earlier than by PCR. Phylogenetic analysis suggested recombination or superinfection in one patient. Deep sequencing of HCMV enriched from clinical samples excluded resistance in 7 of 11 subjects and identified resistance mutations earlier than conventional PCR-based resistance testing in 2 patients. Detection of multiple low level resistance mutations was associated with poor outcome

Implementation und Nutzung von Unterrichtsmaterialien im schulischen Unterricht – Eine Bestandsaufnahme der mathematisch-naturwissenschaftlichen Fächer

Die Bereitstellung von Unterrichtsmaterialien ist eine zentrale Strategie, die professionelle Weiterbildung von Lehrkräften zu unterstützen und die Qualität von Unterricht zu erhöhen. Allerdings werden viele empirisch fundierte Materialien gegenwärtig kaum an Schulen eingesetzt. Eine Analyse von knapp 50 Studien zur Nutzung von Unterrichtsmaterialien impliziert, dass Unterrichtsmaterialien zwar ein großes Potential zur Verbesserung der Unterrichtsqualität aufweisen, ermöglicht aber auch die Identifizierung von Hinderungsfaktoren der Implementation solcher Materialien. Auf Grundlage dieser Ergebnisse werden Empfehlungen für zukünftige Forschungs- und Entwicklungsprojekte abgeleitet, mit dem Ziel langfristig die Umsetzung innovativer Unterrichtskonzepte und darauf abgestimmter Materialien an Schulen zu erhöhen

Implementation fachdidaktischer Innovation am Beispiel des Münchener Unterrichtskonzepts zur Quantenmechanik

Zu den Aufgaben fachdidaktischer Forschung gehört die Verbesserung von Lehr-Lern-Prozessen auf Grundlage von empirischen Forschungsergebnissen. Ein möglicher Weg, solche Innovationen an Schulen zu implementieren, verläuft über die Bereitstellung von Unterrichtsmaterialien. Allerdings liegen bisher nur wenige Erkenntnisse zu ihrer Nutzung vor.Ziel der vorgestellten Studie ist es daher, mehr über die Wirkmechanismen bei der Implementation von Unterrichtsmaterialien herauszufinden. Dazu werden zehn Lehrkräfte, welche das Münchener Unterrichtskonzept zur Quantenmechanik zur Verfügung gestellt bekommen, im Unterricht begleitet. In zwei Interviews zu Beginn und am Ende einer Unterrichtsreihe zur Quantenmechanik werden mögliche Einflussfaktoren der Materialnutzung erfragt. Um zudem die Implementation genauer zu untersuchen, werden drei Unterrichtsstunden videographiert und durch Stimulated Recall-Interviews Hintergründe zu den Entscheidungen der Lehrkräfte abgefragt.Aus den Ergebnissen der Pilotierungsinterviews lässt sich ableiten, dass Lehrkräfte oftmals nur Teile des bereitgestellten Konzepts umsetzen

Implementation fachdidaktischer Innovation am Beispiel des Münchener Unterrichtskonzepts zur Quantenmechanik

Zu den Aufgaben fachdidaktischer Forschung gehört die Verbesserung von Lehr-Lern-Prozessen auf Grundlage von empirischen Forschungsergebnissen. Ein möglicher Weg, solche Innovationen an Schulen zu implementieren, verläuft über die Bereitstellung von Unterrichtsmaterialien. Allerdings liegen bisher nur wenige Erkenntnisse zu ihrer Nutzung vor.Ziel der vorgestellten Studie ist es daher, mehr über die Wirkmechanismen bei der Implementation von Unterrichtsmaterialien herauszufinden. Dazu werden zehn Lehrkräfte, welche das Münchener Unterrichtskonzept zur Quantenmechanik zur Verfügung gestellt bekommen, im Unterricht begleitet. In zwei Interviews zu Beginn und am Ende einer Unterrichtsreihe zur Quantenmechanik werden mögliche Einflussfaktoren der Materialnutzung erfragt. Um zudem die Implementation genauer zu untersuchen, werden drei Unterrichtsstunden videographiert und durch Stimulated Recall-Interviews Hintergründe zu den Entscheidungen der Lehrkräfte abgefragt.Aus den Ergebnissen der Pilotierungsinterviews lässt sich ableiten, dass Lehrkräfte oftmals nur Teile des bereitgestellten Konzepts umsetzen

A Variant Allele in Varicella-Zoster Virus Glycoprotein B Selected during Production of the Varicella Vaccine Contributes to Its Attenuation

Attenuation of the live varicella Oka vaccine (vOka) has been attributed to mutations in the genome acquired during cell culture passage of pOka (parent strain); however, the precise mechanisms of attenuation remain unknown. Comparative sequence analyses of several vaccine batches showed that over 100 single-nucleotide polymorphisms (SNPs) are conserved across all vaccine batches; 6 SNPs are nearly fixed, suggesting that these SNPs are responsible for attenuation. By contrast, prior analysis of chimeric vOka and pOka recombinants indicates that loci other than these six SNPs contribute to attenuation. Here, we report that pOka consists of a heterogenous population of virus sequences with two nearly equally represented bases, guanine (G) or adenine (A), at nucleotide 2096 of the ORF31 coding sequence, which encodes glycoprotein B (gB) resulting in arginine (R) or glutamine (Q), respectively, at amino acid 699 of gB. By contrast, 2096A/699Q is dominant in vOka (.99.98%). gB699Q/gH/gL showed significantly less fusion activity than gB699R/gH/gL in a cell-based fusion assay. Recombinant pOka with gB669Q (rpOka_gB699Q) had a similar growth phenotype as vOka during lytic infection in cell culture including human primary skin cells; however, rpOka_gB699R showed a growth phenotype similar to pOka. rpOka_gB699R entered neurons from axonal terminals more efficiently than rpOka_gB699Q in the presence of cell membrane-derived vesicles containing gB. Strikingly, when a mixture of pOka with both alleles equally represented was used to infect human neurons from axon terminals, pOka with gB699R was dominant for virus entry. These results identify a variant allele in gB that contributes to attenuation of vOka. IMPORTANCE The live-attenuated varicella vaccine has reduced the burden of chickenpox. Despite its development in 1974, the molecular basis for its attenuation is still not well understood. Since the live-attenuated varicella vaccine is the only licensed human herpesvirus vaccine that prevents primary disease, it is important to understand the mechanism for its attenuation. Here we identify that a variant allele in glycoprotein B (gB) selected during generation of the varicella vaccine contributes to its attenuation. This variant is impaired for fusion, virus entry into neurons from nerve terminals, and replication in human skin cells. Identification of a variant allele in gB, one of the essential herpesvirus core genes, that contributes to its attenuation may provide insights that assist in the development of other herpesvirus vaccines

Prevalence of and factors associated with herpes zoster in England: a cross-sectional analysis of the Health Survey for England

BACKGROUND: Herpes zoster (commonly called shingles) is caused by the reactivation of varicella zoster virus, and results in substantial morbidity. While the risk of zoster increases significantly with age and immunosuppression, relatively little is known about other risk factors for zoster. Moreover, much evidence to date stems from electronic healthcare or administrative data. Hence, the aim of this study was to explore potential risk factors for herpes zoster using survey data from a nationally-representative sample of the general community-dwelling population in England. METHODS: Data were extracted from the 2015 Health Survey for England, an annual cross-sectional representative survey of households in England. The lifetime prevalence of self-reported herpes zoster was described by age, gender and other socio-demographic factors, health behaviours (physical activity levels, body mass index, smoking status and alcohol consumption) and clinical conditions, including; diabetes, respiratory, digestive and genito-urinary system and mental health disorders. Logistic regression models were then used to identify possible factors associated with shingles, and results were presented as odds ratios with 95% confidence intervals. RESULTS: The lifetime prevalence of shingles among the sample was 11.5% (12.6% among women, 10.3% among men), which increased with age. After adjusting for a range of covariates, increased age, female gender (odds ratio: 1.21; 95%CI: 1.03, 1.43), White ethnic backgrounds (odds ratio: 2.00; 95%CI: 1.40, 2.88), moderate physical activity 7 days per week (odds ratio: 1.29; 95%CI: 1.01, 1.66) and digestive disorders (odds ratio: 1.51; 95%CI: 1.13, 1.51) were each associated with increased odds of having had herpes zoster. CONCLUSIONS: Age, gender, ethnicity and digestive disorders may be risk factors for herpes zoster among a nationally representative sample of adults in England. These potential risk factors and possible mechanisms should be further explored using longitudinal studies