Viral Metagenomics on Blood-Feeding Arthropods as a Tool for Human Disease Surveillance

Surveillance and monitoring of viral pathogens circulating in humans and wildlife, together with the identification of emerging infectious diseases (EIDs), are critical for the prediction of future disease outbreaks and epidemics at an early stage. It is advisable to sample a broad range of vertebrates and invertebrates at different temporospatial levels on a regular basis to detect possible candidate viruses at their natural source. However, virus surveillance systems can be expensive, costly in terms of finances and resources and inadequate for sampling sufficient numbers of different host species over space and time. Recent publications have presented the concept of a new virus surveillance system, coining the terms “flying biological syringes”, “xenosurveillance” and “vector-enabled metagenomics”. According to these novel and promising surveillance approaches, viral metagenomics on engorged mosquitoes might reflect the viral diversity of numerous mammals, birds and humans, combined in the mosquitoes’ blood meal during feeding on the host. In this review article, we summarize the literature on vector-enabled metagenomics (VEM) techniques and its application in disease surveillance in humans. Furthermore, we highlight the combination of VEM and “invertebrate-derived DNA” (iDNA) analysis to identify the host DNA within the mosquito midgut

The virome ofGerman bats: comparing virus discovery approaches

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.Peer Reviewe

One-step selection of Vaccinia virus-binding DNA aptamers by MonoLEX

<p>Abstract</p> <p>Background</p> <p>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.</p> <p>Results</p> <p>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 <it>in vitro </it>infection of Vaccinia virus and other orthopoxviruses in a concentration-dependent manner.</p> <p>Conclusion</p> <p>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.</p

The :envihab - Linking biomedical research and technological innovation for Astronaut health

The DLR Institute of Aerospace Medicine has longtime experience in supporting human spaceflights. Ever since contributing to the Spacelab Program, the Institute has supported the psychological and medical astronaut selection, training, and mission operations. The program is flanked by state-of-the-art ground based biological, medical, and psychological research. The :envihab, derived from the words environment and habitat, is a unique medical research facility operated by the DLR Institute of Aerospace Medicine. Within its eight modules, :envihab houses multi-purpose laboratories and specialized equipment for life science research. Furthermore, :envihab features a high-end research ward accommodating study participants in bedrest studies and sleep investigations among others. The module comprises 12 individual rooms and can be conditioned using normobaric hypoxia or hypercapnia. :envihab offers a fully equipped human short-arm centrifuge equipped with a robotic-controlled ultrasonography device for examinations of cardiovascular parameters during exposure to centrifugation. The 3 Tesla PET-MRI allows for sophisticated structural, functional, and molecular imaging. The Biology Lab consists of four microbiology laboratory rooms, one of them is an ISO class 8 clean room. A large hypobaric chamber is suitable for hypobaric hypoxia, normobaric hypoxia, and hypercapnia studies. Finally, advanced cardiovascular, musculoskeletal, metabolic, ophthalmological, and psychological testing as well as highly controlled dietary support make :envihab a worldwide unique human research facility. Meanwhile, several mechanism-oriented sleep studies as well as bed rest studies up to 60 days have been successfully completed. The Institute supports postflight activities of ESA astronauts – the so-called “Direct Return”. Astronauts, crew surgeon and operational staff can be accommodated. The Crew Quarters are access-controlled for infection control and astronaut privacy. All medical post-flight examinations required according to the Medical Standards for Crewmembers can be performed at the adjacent DLR Flight Medicine Clinic. Pre- and post-flight examinations and measurements can be performed at the same site with the same equipment and the same staff, thus, limiting variability. Similarly, post-flight experiments are conducted by DLR scientists within :envihab. In November 2014, Alexander Gerst was the first astronaut to directly return to Cologne, followed by Andreas Mogensen (Sept. 2015), Timothy Peake (June 2016) and Thomas Pesquet (June 2017). We expect ISS commander Alexander Gerst as the next astronaut hosted at :envihab in 2018

Environmental Risk Factors for Chronic Pancreatitis and Pancreatic Cancer

Chronic pancreatitis has long been thought to be mainly associated with immoderate alcohol consumption. The observation that only ∼10% of heavy drinkers develop chronic pancreatitis not only suggests that other environmental factors, such as tobacco smoke, are potent additional risk factors, but also that the genetic component of pancreatitis is more common than previously presumed. Either disease-causing or protective traits have been indentified for mutations in different trypsinogen genes, the gene for the trypsin inhibitor SPINK1, chymotrypsinogen C, and the cystic fibrosis transmembane conductance regulator (CFTR). Other factors that have been proposed to contribute to pancreatitis are obesity, diets high in animal protein and fat, as well as antioxidant deficiencies. For the development of pancreatic cancer, preexisting chronic pancreatitis, more prominently hereditary pancreatitis, is a risk factor. The data on environmental risk factors for pancreatic cancer are, with the notable exception of tobacco smoke, either sparse, unconfirmed or controversial. Obesity appears to increase the risk of pancreatic cancer in the West but not in Japan. Diets high in processed or red meat, diets low in fruits and vegetables, phytochemicals such as lycopene and flavonols, have been proposed and refuted as risk or protective factors in different trials. The best established and single most important risk factor for cancer as well as pancreatitis and the one to clearly avoid is tobacco smoke

Comparison of 454 Ultra-Deep Sequencing and Allele-Specific Real-Time PCR with Regard to the Detection of Emerging Drug-Resistant Minor HIV-1 Variants after Antiretroviral Prophylaxis for Vertical Transmission

Background: Pregnant HIV-infected women were screened for the development of HIV-1 drug resistance after implementation of a triple-antiretroviral transmission prophylaxis as recommended by the WHO in 2006. The study offered the opportunity to compare amplicon-based 454 ultra-deep sequencing (UDS) and allele-specific real-time PCR (ASPCR) for the detection of drug-resistant minor variants in the HIV-1 reverse transcriptase (RT). Methods: Plasma samples from 34 Tanzanian women were previously analysed by ASPCR for key resistance mutations in the viral RT selected by AZT, 3TC, and NVP (K70R, K103N, Y181C, M184V, T215Y/F). In this study, the RT region of the same samples was investigated by amplicon-based UDS for resistance mutations using the 454 GS FLX System. Results: Drug-resistant HIV-variants were identified in 69% (20/29) of women by UDS and in 45% (13/29) by ASPCR. The absolute number of resistance mutations identified by UDS was twice that identified by ASPCR (45 vs 24). By UDS 14 of 24 ASPCR-detected resistance mutations were identified at the same position. The overall concordance between UDS and ASPCR was 61.0% (25/41). The proportions of variants quantified by UDS were approximately 2–3 times lower than by ASPCR. Amplicon generation from samples with viral loads below 20,000 copies/ml failed more frequently by UDS compared to ASPCR (limit of detection = 650 copies/ml), resulting in missing or insufficient sequence coverage. Conclusions: Both methods can provide useful information about drug-resistant minor HIV-1 variants. ASPCR has a higher sensitivity than UDS, but is restricted to single resistance mutations. In contrast, UDS is limited by its requirement for high viral loads to achieve sufficient sequence coverage, but the sequence information reveals the complete resistance patterns within the genomic region analysed. Improvements to the UDS limit of detection are in progress, and UDS could then facilitate monitoring of drug-resistant minor variants in the HIV-1 quasispecies

Paramyxovirus Diversity within One Population of Miniopterus fuliginosus Bats in Sri Lanka

Bats are known as typical reservoirs for a number of viruses, including viruses of the family Paramyxoviridae. Representatives of the subfamily Orthoparamyxovirinae are distributed worldwide and can cause mild to fatal diseases when infecting humans. The research on Paramyxoviruses (PMVs) from different bat hosts all over the world aims to understand the diversity, evolution and distribution of these viruses and to assess their zoonotic potential. A high number of yet unclassified PMVs from bats are recorded. In our study, we investigated bat species from the families Rhinolophidae, Hipposiderae, Pteropodidae and Miniopteridae that are roosting sympatrically in the Wavul Galge cave (Koslanda, Sri Lanka). The sampling at three time points (March and July 2018; January 2019) and screening for PMVs with a generic PCR show the presence of different novel PMVs in 10 urine samples collected from Miniopterus fuliginosus. Sequence analysis revealed a high similarity of the novel strains among each other and to other unclassified PMVs collected from Miniopterus bats. In this study, we present the first detection of PMVs in Sri Lanka and the presence of PMVs in the bat species M. fuliginosus for the first time.Peer Reviewe

Primary ChAdOx1 vaccination does not reactivate pre-existing, cross-reactive immunity

Currently available COVID-19 vaccines include inactivated virus, live attenuated virus, mRNA-based, viral vectored and adjuvanted protein-subunit-based vaccines. All of them contain the spike glycoprotein as the main immunogen and result in reduced disease severity upon SARS-CoV-2 infection. While we and others have shown that mRNA-based vaccination reactivates pre-existing, cross-reactive immunity, the effect of vector vaccines in this regard is unknown. Here, we studied cellular and humoral responses in heterologous adenovirus-vector-based ChAdOx1 nCOV-19 (AZ; Vaxzeria, AstraZeneca) and mRNA-based BNT162b2 (BNT; Comirnaty, BioNTech/Pfizer) vaccination and compared it to a homologous BNT vaccination regimen. AZ primary vaccination did not lead to measurable reactivation of cross-reactive cellular and humoral immunity compared to BNT primary vaccination. Moreover, humoral immunity induced by primary vaccination with AZ displayed differences in linear spike peptide epitope coverage and a lack of anti-S2 IgG antibodies. Contrary to primary AZ vaccination, secondary vaccination with BNT reactivated pre-existing, cross-reactive immunity, comparable to homologous primary and secondary mRNA vaccination. While induced anti-S1 IgG antibody titers were higher after heterologous vaccination, induced CD4(+) T cell responses were highest in homologous vaccinated. However, the overall TCR repertoire breadth was comparable between heterologous AZ-BNT-vaccinated and homologous BNT-BNT-vaccinated individuals, matching TCR repertoire breadths after SARS-CoV-2 infection, too. The reasons why AZ and BNT primary vaccination elicits different immune response patterns to essentially the same antigen, and the associated benefits and risks, need further investigation to inform vaccine and vaccination schedule development

Untersuchung und Eindämmung eines SARS-CoV-2-Alpha-Ausbruchs in einer Pflegeeinrichtung im Landkreis Dithmarschen, Juni 2021

Bewohnende und Personal in Pflegeeinrichtungen gehören zu den Personengruppen, die ab dem 27.12.2020 in Deutschland priorisiert die COVID-19-Impfung erhielten. Diese zeigte eine gute Wirksamkeit hinsichtlich des Schutzes vor Infektionen mit der Alpha-Variante und eine sehr hohe Wirksamkeit vor schweren Krankheitsverläufen. In einer Pflegeeinrichtung im Landkreis Dithmarschen kam es zwischen dem 01.04. und 23.06.2021 dennoch zu einem SARS-CoV-2-Ausbruch, bei dem eine hohe Anzahl vollständig geimpfter Personen infiziert wurde und teilweise schwer erkrankte oder verstarb. Beschrieben werden zum einen die Methoden, die bei der Ausbruchsuntersuchung im Pflegeheim angewandt wurden, und zum anderen die Maßnahmen, die schlussendlich zur Ausbruchseindämmung beitrugen.Peer Reviewe

Contribution of Streptococcus anginosus to Infections Caused by Groups C and G Streptococci, Southern India

This neglected pathogen causes a large portion of these infections