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In November 2011, a new virus of the genus Orthobunyavirus was isolated from diseased cattle in Germany and was provisionally called Schmallenberg virus (SBV)(1). It has caused disease in ruminants, [...]

Abstract Background Previous controlled studies on the effect of non-pharmaceutical interventions (NPI) - namely the use of facemasks and intensified hand hygiene - in preventing household transmission of influenza have not produced definitive results. We aimed to investigate efficacy, acceptability, and tolerability of NPI in households with influenza index patients. Methods We conducted a cluster randomized controlled trial during the pandemic season 2009/10 and the ensuing influenza season 2010/11. We included households with an influenza positive index case in the absence of further respiratory illness within the preceding 14 days. Study arms were wearing a facemask and practicing intensified hand hygiene (MH group), wearing facemasks only (M group) and none of the two (control group). Main outcome measure was laboratory confirmed influenza infection in a household contact. We used daily questionnaires to examine adherence and tolerability of the interventions. Results We recruited 84 households (30 control, 26 M and 28 MH households) with 82, 69 and 67 household contacts, respectively. In 2009/10 all 41 index cases had a influenza A (H1N1) pdm09 infection, in 2010/11 24 had an A (H1N1) pdm09 and 20 had a B infection. The total secondary attack rate was 16% (35/218). In intention-to-treat analysis there was no statistically significant effect of the M and MH interventions on secondary infections. When analysing only households where intervention was implemented within 36 h after symptom onset of the index case, secondary infection in the pooled M and MH groups was significantly lower compared to the control group (adjusted odds ratio 0.16, 95% CI, 0.03-0.92). In a per-protocol analysis odds ratios were significantly reduced among participants of the M group (adjusted odds ratio, 0.30, 95% CI, 0.10-0.94). With the exception of MH index cases in 2010/11 adherence was good for adults and children, contacts and index cases. Conclusions Results suggest that household transmission of influenza can be reduced by the use of NPI, such as facemasks and intensified hand hygiene, when implemented early and used diligently. Concerns about acceptability and tolerability of the interventions should not be a reason against their recommendation. Trial registration The study was registered with ClinicalTrials.gov (Identifier NCT00833885).

Abstract Background Despite the successful eradication of smallpox by the WHO-led vaccination programme, pox virus infections remain a considerable health threat. The possible use of smallpox as a bioterrorism agent as well as the continuous occurrence of zoonotic pox virus infections document the relevance to deepen the understanding for virus host interactions. Since the permissiveness of pox infections is independent of hosts surface receptors, but correlates with the ability of the virus to infiltrate the antiviral host response, it directly depends on the hosts proteome set. In this report the proteome of HEK293 cells infected with Vaccinia Virus strain IHD-W was analyzed by 2-dimensional gel electrophoresis and MALDI-PSD-TOF MS in a bottom-up approach. Results The cellular and viral proteomes of VACV IHD-W infected HEK293 cells, UV-inactivated VACV IHD-W-treated as well as non-infected cells were compared. Derivatization of peptides with 4-sulfophenyl isothiocyanate (SPITC) carried out on ZipTipμ-C18 columns enabled protein identification via the peptides' primary sequence, providing improved s/n ratios as well as signal intensities of the PSD spectra. The expression of more than 24 human proteins was modulated by the viral infection. Effects of UV-inactivated and infectious viruses on the hosts' proteome concerning energy metabolism and proteins associated with gene expression and protein-biosynthesis were quite similar. These effects might therefore be attributed to virus entry and virion proteins. However, the modulation of proteins involved in apoptosis was clearly correlated to infectious viruses. Conclusions The proteome analysis of infected cells provides insight into apoptosis modulation, regulation of cellular gene expression and the regulation of energy metabolism. The confidence of protein identifications was clearly improved by the peptides' derivatization with SPITC on a solid phase support. Some of the identified proteins have not been described in the context of poxvirus infections before and need to be further characterised to identify their meaning for apoptosis modulation and pathogenesis.

To enable rapid and reliable detection of poxviruses in clinical and environmental specimens, a diagnostic approach was developed to detect [less than or equal to] 3 PFU of infectious poxvirus [...]

Background: The reliable detection of SARS-CoV-2 has become one of the most important contributions to COVID-19 crisis management. With the publication of the first sequences of SARS-CoV-2, several diagnostic PCR assays have been developed and published. In addition to in-house assays the market was flooded with numerous commercially available ready-to-use PCR kits, with both approaches showing alarming shortages in reagent supply. Aim: Here we present a resource-efficient in-house protocol for the PCR detection of SARS-CoV-2 RNA in patient specimens (RKI/ZBS1 SARS-CoV-2 protocol). Methods: Two duplex one-step real-time RT-PCR assays are run simultaneously and provide information on two different SARS-CoV-2 genomic regions. Each one is duplexed with a control that either indicates potential PCR inhibition or proves the successful extraction of nucleic acid from the clinical specimen. Results: Limit of RNA detection for both SARS-CoV-2 assays is below 10 genomes per reaction. The protocol enables testing specimens in duplicate across the two different SARS-CoV-2 PCR assays, saving reagents by increasing testing capacity. The protocol can be run on various PCR cyclers with several PCR master mix kits. Conclusion: The presented RKI/ZBS1 SARS-CoV-2 protocol represents a cost-effective alternative in times of shortages when commercially available ready-to-use kits may not be available or affordable. [ABSTRACT FROM AUTHOR]

The cascade of innovations in biotechnology opens new pathways for biological warfare. The international laboratory network being developed under the UN Secretary-General's Mechanism could provide vital evidence in case of an alleged biological attack. [ABSTRACT FROM AUTHOR]

Abstract Targeting the highly conserved herpes DNA polymerase (DPOL) gene with PCR using panherpes degenerate primers is a powerful tool to universally detect unknown herpesviruses. However, vertebrate hosts are often infected with more than one herpesvirus in the same tissue, and pan-herpes DPOL PCR often favors the amplification of one viral sequence at the expense of the others. Here we present two different technical approaches that overcome this obstacle: (i) Pan-herpes DPOL PCR is carried out in the presence of an oligonucleotide substituted with locked nucleic acids (LNA).This suppresses the amplification of a specific herpesvirus DPOL sequence by a factor of approximately 1000, thereby enabling the amplification of a second, different DPOL sequence. (ii) The less conserved glycoprotein B (gB) gene is targeted with several sets of degenerate primers that are restricted to gB genes of different herpesvirus subfamilies or genera. These techniques enable the amplification of gB and DPOL sequences of multiple viruses from a single specimen. The partial gB and DPOL sequences can be connected by long-distance PCR, producing final contiguous sequences of approximately 3.5 kbp. Such sequences include parts of two genes and therefore allow for a robust phylogenetic analysis. To illustrate this principle, six novel herpesviruses of the genera Rhadinovirus, Lymphocryptovirus and Cytomegalovirus were discovered in multi-infected samples of non-human primates and phylogenetically characterized.

Abstract Background As a new class of therapeutic and diagnostic reagents, more than fifteen years ago RNA and DNA aptamers were identified as binding molecules to numerous small compounds, proteins and rarely even to complete pathogen particles. Most aptamers were isolated from complex libraries of synthetic nucleic acids by a process termed SELEX based on several selection and amplification steps. Here we report the application of a new one-step selection method (MonoLEX) to acquire high-affinity DNA aptamers binding Vaccinia virus used as a model organism for complex target structures. Results The selection against complete Vaccinia virus particles resulted in a 64-base DNA aptamer specifically binding to orthopoxviruses as validated by dot blot analysis, Surface Plasmon Resonance, Fluorescence Correlation Spectroscopy and real-time PCR, following an aptamer blotting assay. The same oligonucleotide showed the ability to inhibit in vitro infection of Vaccinia virus and other orthopoxviruses in a concentration-dependent manner. Conclusion The MonoLEX method is a straightforward procedure as demonstrated here for the identification of a high-affinity DNA aptamer binding Vaccinia virus. MonoLEX comprises a single affinity chromatography step, followed by subsequent physical segmentation of the affinity resin and a single final PCR amplification step of bound aptamers. Therefore, this procedure improves the selection of high affinity aptamers by reducing the competition between aptamers of different affinities during the PCR step, indicating an advantage for the single-round MonoLEX method.

Abstract Background Stem cell transplantation as therapy for hematological disorders is often hampered by severe graft-versus-host-disease. This may be reduced by umbilical cord blood transplantation, an effect that has been attributed to qualitative differences between neonatal and adult T cells. We compared levels of secreted proteins and cytokine mRNA induced in cord blood leukocytes (CBL) and adult blood leukocytes (ABL) by various stimuli. Results While interleukin-2 (IL-2) levels were similar in CBL and ABL, there was less induction of the Th1 cytokine interferon-γ in CBL. Production of the Th2 cytokines IL-4, IL-5, and IL-13 and the hematopoietic cytokine IL-3 was much lower in CBL versus ABL after T-cell receptor-mediated stimulation, whereas production of GM-CSF was comparable in the 2 cell types. The lower levels of Th1 and Th2 cytokines were maintained in CBL during a 4-day time-course study, while after 12 hours IL-3 and GM-CSF reached in CBL levels similar to those in ABL. For all cytokines except IFNγ, the IC50 values for inhibition by cyclosporin A were similar in ABL and CBL. In contrast, there was less expression and activation of transcription factors in CBL. Activation of NF-κB by TPA/ionomycin was detected in ABL but not CBL. Furthermore, there was less expression of the Th subset-specific transcription factors T-bet and c-maf in CBL versus ABL, whereas GATA-3 expression was similar. Expression of T-bet and c-maf correlated with expression of the Th1 and Th2 cytokines, respectively. Time course experiments revealed that T-bet expression was stimulated in both cell types, whereas c-maf and GATA-3 were induced only in ABL. Conclusion The diminished capability of CBL to synthesize cytokines is probably due to decreased activation of NF-κB, whereas differences in Th subsets are due to differences in regulation of Th lineage-specific transcriptions factors. We propose that the reduced incidence and severity of GvHD after allogeneic transplantation of umbilical CB cells is due to lesser activation of specific transcription factors and a subsequent reduction in production of certain cytokines.

Abstract Human herpesvirus-6 (HHV-6) can infect blood cells and thereby may inhibit hematopoietic stem and progenitor cell expansion and differentiation. In this context, it has been discussed if early progenitor cells can be infected by HHV-6. CD46 was identified as one possible cellular surface receptor for HHV-6. The study presented here had been done to get insight into the susceptibility of various leukocyte subpopulations to HHV-6 (including early hematopoietic progenitors) by determining the amount of CD46 molecules expressed on their surfaces. Human cord blood cells, peripheral blood cells and G-CSF mobilised progenitor cells were analysed by flow cytometry. CD46 molecule number per cell was determined and compared to calibration beads conjugated with known ratio of PE per bead. Highest CD46 expression was detected on B- lymphocytes, whereas T-lymphocytes only showed about half of the amount found on B cells. Hematopoietic progenitors also carried CD46 at intermediate levels. Unexpectedly, CD46 expression on progenitors from G-CSF mobilised leukapheresis products was approximately 20% of that found on comparable cells from untreated cord blood. In conclusion, hematopoietic progenitor cells express CD46 on their surface, thereby fulfilling a basic requirement for the susceptibility of HHV-6 infection.

Bats are known to be reservoirs of several highly pathogenic viruses. Hence, the interest in bat virus discovery has been increasing rapidly over the last decade. So far, most studies have focused on a single type of virus detection method, either PCR, virus isolation or virome sequencing. Here we present a comprehensive approach in virus discovery, using all three discovery methods on samples from the same bats. By family-specific PCR screening we found sequences of paramyxoviruses, adenoviruses, herpesviruses and one coronavirus. By cell culture we isolated a novel bat adenovirus and bat orthoreovirus. Virome sequencing revealed viral sequences of ten different virus families and orders: three bat nairoviruses, three phenuiviruses, one orbivirus, one rotavirus, one orthoreovirus, one mononegavirus, five parvoviruses, seven picornaviruses, three retroviruses, one totivirus and two thymoviruses were discovered. Of all viruses identified by family-specific PCR in the original samples, none was found by metagenomic sequencing. Vice versa, none of the viruses found by the metagenomic virome approach was detected by family-specific PCRs targeting the same family. The discrepancy of detected viruses by different detection approaches suggests that a combined approach using different detection methods is necessary for virus discovery studies. [ABSTRACT FROM AUTHOR]

Copyright of Bundesgesundheitsblatt - Gesundheitsforschung - Gesundheitsschutz is the property of Springer Nature and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Copyright of Bundesgesundheitsblatt - Gesundheitsforschung - Gesundheitsschutz is the property of Springer Nature and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

SARS-CoV-2 is the causative of the COVID-19 disease, which has spread pandemically around the globe within a few months. It is therefore necessary to collect fundamental information about the disease, its epidemiology and treatment, as well as about the virus itself. While the virus has been identified rapidly, detailed ultrastructural analysis of virus cell biology and architecture is still in its infancy. We therefore studied the virus morphology and morphometry of SARS-CoV-2 in comparison to SARS-CoV as it appears in Vero cell cultures by using conventional thin section electron microscopy and electron tomography. Both virus isolates, SARS-CoV Frankfurt 1 and SARS-CoV-2 Italy-INMI1, were virtually identical at the ultrastructural level and revealed a very similar particle size distribution with a median of about 100 nm without spikes. Maximal spike length of both viruses was 23 nm. The number of spikes per virus particle was about 30% higher in the SARS-CoV than in the SARS-CoV-2 isolate. This result complements a previous qualitative finding, which was related to a lower productivity of SARS-CoV-2 in cell culture in comparison to SARS-CoV. [ABSTRACT FROM AUTHOR]

Abstract Ten potential reference genes were compared for their use in experiments investigating cellular mRNA expression of virus infected cells. Human cell lines were infected with Cytomegalovirus, Human Herpesvirus-6, Camelpox virus, SARS coronavirus or Yellow fever virus. The expression levels of these genes and the viral replication were determined by real-time PCR. Genes were ranked by the BestKeeper tool, the GeNorm tool and by criteria we reported previously. Ranking lists of the genes tested were tool dependent. However, over all, β-actin is an unsuitable as reference gene, whereas TATA-Box binding protein and peptidyl-prolyl-isomerase A are stable reference genes for expression studies in virus infected cells.

Abstract Background Prompt laboratory diagnosis of Herpes simplex virus (HSV) infection facilitates patient management and possible initiation of antiviral therapy. In our laboratory, which receives various specimen types for detection of HSV, we use enzyme immunoassay (EIA) for rapid detection and culture of this virus. The culture of HSV has traditionally been accepted as the diagnostic 'gold standard'. In this study, we compared the use of real time PCR (LightCycler) for amplification, detection and subtyping of specific DNA with our in-house developed rapid and culture tests for HSV. Results The LightCycler PCR (LC-PCR) detected and subtyped HSV in 99% (66/67) of HSV positive specimens, compared to 81% (54/67) by rapid antigen EIA or 57% (36/63) by culture. A specimen was considered positive when two or more tests yielded HSV identifications or was culture positive. Discordant results were confirmed with an in-house developed PCR-ELISA or DNA sequence analysis. The typing results obtained with the LC-PCR and by culture amplified test were completely concordant. Conclusions This study showed that the LC-PCR provided a highly sensitive test for simultaneous detection and subtyping of HSV in a single reaction tube. In addition to increased sensitivity, the LightCycler PCR provided reduced turn-around-times (2 hours) when compared to enzyme immunoassay (4 hours) or culture (4 days).

Bats have been gaining attention as potential reservoir hosts of numerous viruses pathogenic to animals and man. Issyk-Kul virus, a member of the family Nairoviridae, was first isolated in the 1970s from vespertilionid bats in Central Asia. Issyk-Kul virus has been described as human-pathogenic virus, causing febrile outbreaks in humans with headaches, myalgia and nausea. Here we describe the detection of a novel strain of Issyk-Kul virus from Eptesicus nilssonii in Germany. This finding indicates for the first time the prevalence of these zoonotic viruses in Europe. [ABSTRACT FROM AUTHOR]

Bacterial proteins dubbed virulence factors (VFs) are a highly diverse group of sequences, whose only obvious commonality is the very property of being, more or less directly, involved in virulence. It is therefore tempting to speculate whether their prediction, based on direct sequence similarity (seqsim) to known VFs, could be enhanced or even replaced by using machine-learning methods. Specifically, when trained on a large and diverse set of VFs, such may be able to detect putative, non-trivial characteristics shared by otherwise unrelated VF families and therefore better predict novel VFs with insignificant similarity to each individual family. We therefore first reassess the performance of dimer-based Support Vector Machines, as used in the widely used MP3 method, in light of seqsim-only and seqsim/dimer-hybrid classifiers. We then repeat the analysis with a novel, considerably more diverse data set, also addressing the important problem of negative data selection. Finally, we move on to the real-world use case of proteome-wide VF prediction, outlining different approaches to estimating specificity in this scenario. We find that direct seqsim is of unparalleled importance and therefore should always be exploited. Further, we observe strikingly low correlations between different feature and classifier types when ranking proteins by VF likeness. We therefore propose a 'best of each world' approach to prioritize proteins for experimental testing, focussing on the top predictions of each classifier. Further, classifiers for individual VF families should be developed. [ABSTRACT FROM AUTHOR]

Sarcocystis scandentiborneensis sp. nov. was discovered in histological sections of striated musculature of treeshrews (Tupaia minor , T. tana) from Northern Borneo. Sarcocysts were cigar-shaped, 102 μm–545 μm long, and on average 53 μm in diameter. The striated cyst wall varied in thickness (2–10 μm), depending on whether the finger-like, villous protrusions (VP) were bent. Ultrastructurally, sarcocysts were similar to wall type 12 but basal microtubules extended into VPs that tapered off with a unique U-shaped, electron-dense apical structure. In phylogenetic trees of the nuclear 18S rRNA gene, S. scandentiborneensis formed a distinct branch within a monophyletic subclade of Sarcocystis spp. with (colubrid) snake-rodent life cycle. We mapped all intraspecific (two haplotypes) and interspecific nucleotide substitutions to the secondary structure of the 18S rRNA gene: in both cases, the highest variability occurred within helices V2 and V4 but intraspecific variability mostly related to transitions, while transition/transversion ratios between S. scandentiborneensis , S. zuoi , and S. clethrionomyelaphis were skewed towards transversions. Lack of relevant sequences restricted phylogenetic analysis of the mitochondrial Cytochrome C oxidase subunit I (COI) gene to include only one species of Sarcocystis recovered from a snake host (S. pantherophisi) with which the new species formed a sister relationship. We confirm the presence of the functionally important elements of the COI barcode amino acid sequence of S. scandentiborneensis , whereby the frequency of functionally important amino acids (Alanine, Serine) was markedly different to other taxa of the Sarcocystidae. We regard S. scandentiborneensis a new species, highlighting that structurally or functionally important aspects of the 18S rRNA and COI could expand their utility for delineation of species. We also address the question why treeshrews, believed to be close to primates, carry a parasite that is genetically close to a Sarcocystis lineage preferably developing in the Rodentia as intermediate hosts. Image 1 • Sarcocystis scandentiborneensis sp. nov. identified from Tupaia tana and T. minor. • Putative snake-treeshrew life cycle. • Unique brush-like cyst wall with an electron-dense apical structure. • Transition/transversion ratios of 18S rDNA improve species discrimination. • COI: Interspecific differences of functionally important amino acids of barcode area. [ABSTRACT FROM AUTHOR]