Rapid Isothermal Detection of Pathogenic Clostridioides difficile Using Recombinase Polymerase Amplification.

Nosocomial-associated diarrhea due to Clostridioides difficile infection (CDI) is diagnosed after sample precultivation by the detection of the toxins in enzyme immunoassays or via toxin gene nucleic acid amplification. Rapid and direct diagnosis is important for targeted treatment to prevent severe cases and recurrence. We developed two singleplex and a one-pot duplex fluorescent 15 min isothermal recombinase polymerase amplification (RPA) assays targeting the toxin genes A and B (tcdA and tcdB). Furthermore, we adapted the singleplex RPA to a 3D-printed microreactor device. Analytical sensitivity was determined using a DNA standard and DNA extracts of 20 C. difficile strains with different toxinotypes. Nineteen clostridial and gastrointestinal bacteria strains were used to determine analytical specificity. Adaptation of singleplex assays to duplex assays in a 50 µL volume required optimized primer and probe concentrations. A volume reduction by one-fourth (12.4 µL) was established for the 3D-printed microreactor. Mixing of RPA was confirmed as essential for optimal analytical sensitivity. Detection limits (LOD) ranging from 119 to 1411 DNA molecules detected were similar in the duplex tube format and in the singleplex 3D-printed microreactor format. The duplex RPA allows the simultaneous detection of both toxins important for the timely and reliable diagnosis of CDI. The 3D-printed reaction chamber can be developed into a microfluidic lab-on-a-chip system use at the point of care.

Examination of blood samples using deep learning and mobile microscopy.

BACKGROUND: Microscopic examination of human blood samples is an excellent opportunity to assess general health status and diagnose diseases. Conventional blood tests are performed in medical laboratories by specialized professionals and are time and labor intensive. The development of a point-of-care system based on a mobile microscope and powerful algorithms would be beneficial for providing care directly at the patient's bedside. For this purpose human blood samples were visualized using a low-cost mobile microscope, an ocular camera and a smartphone. Training and optimisation of different deep learning methods for instance segmentation are used to detect and count the different blood cells. The accuracy of the results is assessed using quantitative and qualitative evaluation standards. RESULTS: Instance segmentation models such as Mask R-CNN, Mask Scoring R-CNN, D2Det and YOLACT were trained and optimised for the detection and classification of all blood cell types. These networks were not designed to detect very small objects in large numbers, so extensive modifications were necessary. Thus, segmentation of all blood cell types and their classification was feasible with great accuracy: qualitatively evaluated, mean average precision of 0.57 and mean average recall of 0.61 are achieved for all blood cell types. Quantitatively, 93% of ground truth blood cells can be detected. CONCLUSIONS: Mobile blood testing as a point-of-care system can be performed with diagnostic accuracy using deep learning methods. In the future, this application could enable very fast, cheap, location- and knowledge-independent patient care.

Does Being Ill Improve Acceptance of Medical Technology?-A Patient Survey with the Technology Usage Inventory.

Acceptance of new medical technology may be influenced by social conditions and an individual's background and particular situation. We studied this acceptance by hypothesizing that current and former COVID-19 patients would be more likely to accept an electrocardiogram (ECG) "patch" (attached to the chest) that allows continuous monitoring of the heart than individuals who did not have the disease and thus the respective experience. Currently infected COVID-19 patients, individuals who had recovered from COVID-19, and a control group were recruited online through Facebook (and Instagram) and through general practitioners (GPs). Demographic information and questions tailored to the problem were collected via an online questionnaire. An online survey was chosen in part because of the pandemic conditions, and Facebook was chosen because of the widespread discussions of health topics on that platform. The results confirmed the central hypothesis that people who had experienced a disease are more willing to accept new medical technologies and showed that curiosity about new technologies and willingness to use them were significantly higher in the two groups currently or previously affected by COVID-19, whereas fears of being "monitored" (in the sense of surveillance) were significantly higher among people who had not experienced the disease and threat. Experiencing a serious disease ("patient experience") promotes acceptance of new medical technologies.

Suitcase Lab for Rapid Detection of SARS-CoV-2 Based on Recombinase Polymerase Amplification Assay.

In March 2020, the SARS-CoV-2 virus outbreak was declared as a world pandemic by the World Health Organization (WHO). The only measures for controlling the outbreak are testing and isolation of infected cases. Molecular real-time polymerase chain reaction (PCR) assays are very sensitive but require highly equipped laboratories and well-trained personnel. In this study, a rapid point-of-need detection method was developed to detect the RNA-dependent RNA polymerase (RdRP), envelope protein (E), and nucleocapsid protein (N) genes of SARS-CoV-2 based on the reverse transcription recombinase polymerase amplification (RT-RPA) assay. RdRP, E, and N RT-RPA assays required approximately 15 min to amplify 2, 15, and 15 RNA molecules of molecular standard/reaction, respectively. RdRP and E RT-RPA assays detected SARS-CoV-1 and 2 genomic RNA, whereas the N RT-RPA assay identified only SARS-CoV-2 RNA. All established assays did not cross-react with nucleic acids of other respiratory pathogens. The RT-RPA assay's clinical sensitivity and specificity in comparison to real-time RT-PCR (n = 36) were 94 and 100% for RdRP; 65 and 77% for E; and 83 and 94% for the N RT-RPA assay. The assays were deployed to the field, where the RdRP RT-RPA assays confirmed to produce the most accurate results in three different laboratories in Africa (n = 89). The RPA assays were run in a mobile suitcase laboratory to facilitate the deployment at point of need. The assays can contribute to speed up the control measures as well as assist in the detection of COVID-19 cases in low-resource settings.

3D Printed Monolithic Microreactors for Real-Time Detection of Klebsiella pneumoniae and the Resistance Gene blaNDM-1 by Recombinase Polymerase Amplification.

We investigate the compatibility of three 3D printing materials towards real-time recombinase polymerase amplification (rtRPA). Both the general ability of the rtRPA reaction to occur while in contact with the cured 3D printing materials as well as the residual autofluorescence and fluorescence drift in dependence on post curing of the materials is characterized. We 3D printed monolithic rtRPA microreactors and subjected the devices to different post curing protocols. Residual autofluorescence and drift, as well as rtRPA kinetics, were then measured in a custom-made mobile temperature-controlled fluorescence reader (mTFR). Furthermore, we investigated the effects of storage on the devices over a 30-day period. Finally, we present the single- and duplex rtRPA detection of both the organism-specific Klebsiella haemolysin (khe) gene and the New Delhi metallo-ß-lactamase 1 (blaNDM-1) gene from Klebsiella pneumoniae. Results: No combination of 3D printing resin and post curing protocol completely inhibited the rtRPA reaction. The autofluorescence and fluorescence drift measured were found to be highly dependent on printing material and wavelength. Storage had the effect of decreasing the autofluorescence of the investigated materials. Both khe and blaNDM-1 were successfully detected by single- and duplex-rtRPA inside monolithic rtRPA microreactors printed from NextDent Ortho Clear (NXOC). The reaction kinetics were found to be close to those observed for rtRPA performed in a microcentrifuge tube without the need for mixing during amplification. Singleplex assays for both khe and blaNDM-1 achieved a limit of detection of 2.5 × 101 DNA copies while the duplex assay achieved 2.5 × 101 DNA copies for khe and 2.5 × 102 DNA copies for blaNDM-1. Impact: We expand on the state of the art by demonstrating a technology that can manufacture monolithic microfluidic devices that are readily suitable for rtRPA. The devices exhibit very low autofluorescence and fluorescence drift and are compatible with RPA chemistry without the need for any surface pre-treatment such as blocking with, e.g., BSA or PEG.

Rapid Detection of SARS-CoV-2 by Low Volume Real-Time Single Tube Reverse Transcription Recombinase Polymerase Amplification Using an Exo Probe with an Internally Linked Quencher (Exo-IQ).

BACKGROUND: The current outbreak of SARS-CoV-2 has spread to almost every country with more than 5 million confirmed cases and over 300,000 deaths as of May 26, 2020. Rapid first-line testing protocols are needed for outbreak control and surveillance. METHODS: We used computational and manual designs to generate a suitable set of reverse transcription recombinase polymerase amplification (RT-RPA) primer and exonuclease probe, internally quenched (exo-IQ), sequences targeting the SARS-CoV-2 N gene. RT-RPA sensitivity was determined by amplification of in vitro transcribed RNA standards. Assay selectivity was demonstrated with a selectivity panel of 32 nucleic acid samples derived from common respiratory viruses. To validate the assay against full-length SARS-CoV-2 RNA, total viral RNA derived from cell culture supernatant and 19 nasopharyngeal swab samples (8 positive and 11 negative for SARS-CoV-2) were screened. All results were compared to established RT-qPCR assays. RESULTS: The 95% detection probability of the RT-RPA assay was determined to be 7.74 (95% CI: 2.87-27.39) RNA copies per reaction. The assay showed no cross-reactivity to any other screened coronaviruses or respiratory viruses of clinical significance. The developed RT-RPA assay produced 100% diagnostic sensitivity and specificity when compared to RT-qPCR (n = 20). CONCLUSIONS: With a run time of 15 to 20 minutes and first results being available in under 7 minutes for high RNA concentrations, the reported assay constitutes one of the fastest nucleic acid based detection methods for SARS-CoV-2 to date and may provide a simple-to-use alternative to RT-qPCR for first-line screening at the point of need.

A lab-on-a-chip for free-flow electrophoretic preconcentration of viruses and gel electrophoretic DNA extraction.

Nucleic acid amplification techniques such as real-time PCR are essential instruments for the identification and quantification of viruses. They are fast, very sensitive and highly specific, but often require elaborate and labor intensive sample preparation to achieve successful amplification of the target sequence. In this work we demonstrate the complete microfluidic preparation of amplifiable virus DNA from dilute specimens. Our approach combines free-flow electrophoretic preconcentration of viral particles with thermal lysis and gel-electrophoretic nucleic acid extraction on a single device. The on-chip preconcentration achieves a capture efficiency of >99% for dilute suspensions of bacteriophage PhiX174. Following preconcentration, phages are thermally lysed and released DNA is recovered after 40 s of on-chip gel-electrophoresis with a recovery rate of ∼73%. Furthermore we demonstrate a detection limit of ∼1 PFU ml-1 (∼0.02 DNA copies per µl) for the detection of bacteriophage PhiX174 by PCR. To simplify operation of the device, we describe the development of a custom-made chip holder as well as a compact peristaltic pump and power supply, which enable user-friendly operation with low risk of cross-contamination and high potential for automation in the field of point-of-care diagnostics.

A lab-on-a-chip for rapid miRNA extraction.

We present a simple to operate microfluidic chip system that allows for the extraction of miRNAs from cells with minimal hands-on time. The chip integrates thermoelectric lysis (TEL) of cells with native gel-electrophoretic elution (GEE) of released nucleic acids and uses non-toxic reagents while requiring a sample volume of only 5 µl. These properties as well as the fast process duration of 180 seconds make the system an ideal candidate to be part of fully integrated point-of-care applications for e.g. the diagnosis of cancerous tissue. GEE was characterized in comparison to state-of-the-art silica column (SC) based RNA recovery using the mirVana kit (Ambion) as a reference. A synthetic miRNA (miR16) as well as a synthetic snoRNA (SNORD48) were subjected to both GEE and SC. Subsequent detection by stem-loop RT-qPCR demonstrated a higher yield for miRNA recovery by GEE. SnoRNA recovery performance was found to be equal for GEE and SC, indicating yield dependence on RNA length. Coupled operation of the chip (TEL + GEE) was characterized using serial dilutions of 5 to 500 MCF7 cancer cells in suspension. Samples were split and cells were subjected to either on-chip extraction or SC. Eluted miRNAs were then detected by stem-loop RT-qPCR without any further pre-processing. The extraction yield from cells was found to be up to ~200-fold higher for the chip system under non-denaturing conditions. The ratio of eluted miRNAs is shown to be dependent on the degree of complexation with miRNA associated proteins by comparing miRNAs purified by GEE from heat-shock and proteinase-K based lysis.

Recombinase polymerase amplification assay for rapid detection of Monkeypox virus.

In this study, a rapid method for the detection of Central and West Africa clades of Monkeypox virus (MPXV) using recombinase polymerase amplification (RPA) assay targeting the G2R gene was developed. MPXV, an Orthopoxvirus, is a zoonotic dsDNA virus, which is listed as a biothreat agent. RPA was operated at a single constant temperature of 42°C and produced results within 3 to 10 minutes. The MPXV-RPA-assay was highly sensitive with a limit of detection of 16 DNA molecules/µl. The clinical performance of the MPXV-RPA-assay was tested using 47 sera and whole blood samples from humans collected during the recent MPXV outbreak in Nigeria as well as 48 plasma samples from monkeys some of which were experimentally infected with MPXV. The specificity of the MPXV-RPA-assay was 100% (50/50), while the sensitivity was 95% (43/45). This new MPXV-RPA-assay is fast and can be easily utilised at low resource settings using a solar powered mobile suitcase laboratory.

Development of Mobile Laboratory for Viral Hemorrhagic Fever Detection in Africa.

Background: A mobile laboratory transportable on commercial flights was developed to enable local response to viral hemorrhagic fever outbreaks. Methods: The development progressed from use of mobile real-time reverse-transcription polymerase chain reaction to mobile real-time recombinase polymerase amplification. In this study, we describe various stages of the mobile laboratory development. Results: A brief overview of mobile laboratory deployments, which culminated in the first on-site detection of Ebola virus disease (EVD) in March 2014, and their successful use in a campaign to roll back EVD cases in Conakry in the West Africa Ebola virus outbreak are described. Conclusions: The developed mobile laboratory successfully enabled local teams to perform rapid disgnostic testing for viral hemorrhagic fever.

Recombinase polymerase amplification assay for rapid detection of lumpy skin disease virus.

BACKGROUND: Lumpy skin disease virus (LSDV) is a Capripoxvirus infecting cattle and Buffalos. Lumpy skin disease (LSD) leads to significant economic losses due to hide damage, reduction of milk production, mastitis, infertility and mortalities (10 %). Early detection of the virus is crucial to start appropriate outbreak control measures. Veterinarians rely on the presence of the characteristic clinical signs of LSD. Laboratory diagnostics including virus isolation, sequencing and real-time polymerase chain reaction (PCR) are performed at well-equipped laboratories. In this study, a portable, simple, and rapid recombinase polymerase amplification (RPA) assay for the detection of LSDV-genome for the use on farms was developed. RESULTS: The LSDV RPA assay was performed at 42 °C and detected down to 179 DNA copies/reaction in a maximum of 15 min. Unspecific amplification was observed with neither LSDV-negative samples (n = 12) nor nucleic acid preparations from orf virus, bovine papular stomatitis virus, cowpoxvirus, Peste des petits ruminants and Blue tongue virus (serotypes 1, 6 and 8). The clinical sensitivity of the LSDV RPA assay matched 100 % (n = 22) to real-time PCR results. In addition, the LSDV RPA assay detected sheep and goat poxviruses. CONCLUSION: The LSDV RPA assay is a rapid and sensitive test that could be implemented in field or at quarantine stations for the identification of LSDV infected case.

Biosafety standards for working with Crimean-Congo hemorrhagic fever virus.

In countries from which Crimean-Congo haemorrhagic fever (CCHF) is absent, the causative virus, CCHF virus (CCHFV), is classified as a hazard group 4 agent and handled in containment level (CL)-4. In contrast, most endemic countries out of necessity have had to perform diagnostic tests under biosafety level (BSL)-2 or -3 conditions. In particular, Turkey and several of the Balkan countries have safely processed more than 100 000 samples over many years in BSL-2 laboratories. It is therefore advocated that biosafety requirements for CCHF diagnostic procedures should be revised, to allow the tests required to be performed under enhanced BSL-2 conditions with appropriate biosafety laboratory equipment and personal protective equipment used according to standardized protocols in the countries affected. Downgrading of CCHFV research work from CL-4, BSL-4 to CL-3, BSL-3 should also be considered.

A Field-Deployable Reverse Transcription Recombinase Polymerase Amplification Assay for Rapid Detection of the Chikungunya Virus.

BACKGROUND: Chikungunya virus (CHIKV) is a mosquito-borne virus currently transmitted in about 60 countries. CHIKV causes acute flu-like symptoms and in many cases prolonged musculoskeletal and joint pain. Detection of the infection is mostly done using RT-RCR or ELISA, which are not suitable for point-of-care diagnosis. METHODOLOGY/PRINCIPAL FINDINGS: In this study, a reverse transcription recombinase polymerase amplification (RT-RPA) assay for the detection of the CHIKV was developed. The assay sensitivity, specificity, and cross-reactivity were tested. CHIKV RT-RPA assay detected down to 80 genome copies/reaction in a maximum of 15 minutes. It successfully identified 18 isolates representing the three CHIKV genotypes. No cross-reactivity was detected to other alphaviruses and arboviruses except O'nyong'nyong virus, which could be differentiated by a modified RPA primer pair. Seventy-eight samples were screened both by RT-RPA and real-time RT-PCR. The diagnostic sensitivity and specificity of the CHIKV RT-RPA assay were determined at 100%. CONCLUSIONS/SIGNIFICANCE: The developed RT-RPA assay represents a promising method for the molecular detection of CHIKV at point of need.

Diagnostics-in-a-Suitcase: Development of a portable and rapid assay for the detection of the emerging avian influenza A (H7N9) virus.

BACKGROUND: In developing countries, equipment necessary for diagnosis is only available in few central laboratories, which are less accessible and of limited capacity to test large numbers of incoming samples. Moreover, the transport conditions of samples are inadequate, therefore leading to unreliable results. OBJECTIVES: The development of a rapid, inexpensive, and simple test would allow mobile detection of viruses. STUDY DESIGN: A suitcase laboratory "Diagnostics-in-a-Suitcase" (56cm×45.5cm×26.5cm) containing all reagents and devices necessary for performing a reverse transcription recombinase polymerase amplification (RT-RPA) assay was developed. As an example, two RT-RPA assays were established for the detection of hemagglutinin (H) and neuraminidase (N) genes of the novel avian influenza (H7N9) virus. RESULTS: The sensitivities of the H7 and the N9 RT-RPA assays were 10 and 100 RNA molecules, respectively. The assays were performed at a single temperature (42°C). The results were obtained within 2-7min. The H7N9 RT-RPA assays did not show a cross-detection either of any other respiratory viruses affecting humans and/or birds or of the human or chicken genomes. All reagents were used, stored, and transported at ambient temperature, that is, cold chain independent. In addition, the Diagnostics-in-a-Suitcase was operated by a solar-powered battery. CONCLUSIONS: The developed assay protocol and mobile setup performed well. Moreover, it can be easily implemented to perform diagnoses at airports, quarantine stations, or farms for rapid on-site viral nucleic acid detection.

Recombinase Polymerase Amplification Assay for Rapid Diagnostics of Dengue Infection.

BACKGROUND: Over 2.5 billion people are exposed to the risk of contracting dengue fever (DF). Early diagnosis of DF helps to diminish its burden on public health. Real-time reverse transcription polymerase amplification assays (RT-PCR) are the standard method for molecular detection of the dengue virus (DENV). Real-time RT-PCR analysis is not suitable for on-site screening since mobile devices are large, expensive, and complex. In this study, two RT-recombinase polymerase amplification (RT-RPA) assays were developed to detect DENV1-4. METHODOLOGY/PRINCIPAL FINDINGS: Using two quantitative RNA molecular standards, the analytical sensitivity of a RT-RPA targeting the 3´non-translated region of DENV1-4 was found to range from 14 (DENV4) to 241 (DENV1-3) RNA molecules detected. The assay was specific and did not cross detect other Flaviviruses. The RT-RPA assay was tested in a mobile laboratory combining magnetic-bead based total nucleic acid extraction and a portable detection device in Kedougou (Senegal) and in Bangkok (Thailand). In Kedougou, the RT-RPA was operated at an ambient temperature of 38 °C with auxiliary electricity tapped from a motor vehicle and yielded a clinical sensitivity and specificity of 98% (n=31) and 100% (n=23), respectively. While in the field trial in Bangkok, the clinical sensitivity and specificity were 72% (n=90) and 100%(n=41), respectively. CONCLUSIONS/SIGNIFICANCE: During the first 5 days of infection, the developed DENV1-4 RT-RPA assays constitute a suitable accurate and rapid assay for DENV diagnosis. Moreover, the use of a portable fluorescence-reading device broadens its application potential to the point-of-care for outbreak investigations.

Genetic and biological characterization of selected Changuinola viruses (Reoviridae, Orbivirus) from Brazil.

The genus Orbivirus of the family Reoviridae comprises 22 virus species including the Changuinola virus (CGLV) serogroup. The complete genome sequences of 13 CGLV serotypes isolated between 1961 and 1988 from distinct geographical areas of the Brazilian Amazon region were obtained. All viral sequences were obtained from single-passaged CGLV strains grown in Vero cells. CGLVs are the only orbiviruses known to be transmitted by phlebotomine sandflies. Ultrastructure and molecular analysis by electron microscopy and gel electrophoresis, respectively, revealed viral particles with typical orbivirus size and morphology, as well as the presence of a segmented genome with 10 segments. Full-length nucleotide sequencing of each of the ten RNA segments of the 13 CGLV serotypes provided basic information regarding the genome organization, encoded proteins and genetic traits. Segment 2 (encoding VP2) of the CGLV is uncommonly larger in comparison to those found in other orbiviruses and shows varying sizes even among different CGLV serotypes. Phylogenetic analysis support previous serological findings, which indicate that CGLV constitutes a separate serogroup within the genus Orbivirus. In addition, six out of 13 analysed CGLV serotypes showed reassortment of their genome segments.

Evidence for the existence of two new members of the family Chlamydiaceae and proposal of Chlamydia avium sp. nov. and Chlamydia gallinacea sp. nov.

The family Chlamydiaceae with the recombined single genus Chlamydia currently comprises nine species, all of which are obligate intracellular organisms distinguished by a unique biphasic developmental cycle. Anecdotal evidence from epidemiological surveys in flocks of poultry, pigeons and psittacine birds have indicated the presence of non-classified chlamydial strains, some of which may act as pathogens. In the present study, phylogenetic analysis of ribosomal RNA and ompA genes, as well as multi-locus sequence analysis of 11 field isolates were conducted. All independent analyses assigned the strains into two different clades of monophyletic origin corresponding to pigeon and psittacine strains or poultry isolates, respectively. Comparative genome analysis involving the type strains of currently accepted Chlamydiaceae species and the designated type strains representing the two new clades confirmed that the latter could be classified into two different species as their average nucleotide identity (ANI) values were always below 94%, both with the closest relative species and between themselves. In view of the evidence obtained from the analyses, we propose the addition of two new species to the current classification: Chlamydia avium sp. nov. comprising strains from pigeons and psittacine birds (type strain 10DC88(T); DSMZ: DSM27005(T), CSUR: P3508(T)) and Chlamydia gallinacea sp. nov. comprising strains from poultry (type strain 08-1274/3(T); DSMZ: DSM27451(T), CSUR: P3509(T)).

A portable reverse transcription recombinase polymerase amplification assay for rapid detection of foot-and-mouth disease virus.

Foot-and-mouth disease (FMD) is a trans-boundary viral disease of livestock, which causes huge economic losses and constitutes a serious infectious threat for livestock farming worldwide. Early diagnosis of FMD helps to diminish its impact by adequate outbreak management. In this study, we describe the development of a real-time reverse transcription recombinase polymerase amplification (RT-RPA) assay for the detection of FMD virus (FMDV). The FMDV RT-RPA design targeted the 3D gene of FMDV and a 260 nt molecular RNA standard was used for assay validation. The RT-RPA assay was fast (4-10 minutes) and the analytical sensitivity was determined at 1436 RNA molecules detected by probit regression analysis. The FMDV RT-RPA assay detected RNA prepared from all seven FMDV serotypes but did not detect classical swine fever virus or swine vesicular disease virus. The FMDV RT-RPA assay was used in the field during the recent FMD outbreak in Egypt. In clinical samples, reverse transcription polymerase chain reaction (RT-PCR) and RT-RPA showed a diagnostic sensitivity of 100% and 98%, respectively. In conclusion, FMDV RT-RPA was quicker and much easier to handle in the field than real-time RT-PCR. Thus RT-RPA could be easily implemented to perform diagnostics at quarantine stations or farms for rapid spot-of-infection detection.

Molecular phylogeography of tick-borne encephalitis virus in central Europe.

In order to obtain a better understanding of tick-borne encephalitis virus (TBEV) strain movements in central Europe the E gene sequences of 102 TBEV strains collected from 1953 to 2011 at 38 sites in the Czech Republic, Slovakia, Austria and Germany were determined. Bayesian analysis suggests a 350-year history of evolution and spread in central Europe of two main lineages, A and B. In contrast to the east to west spread at the Eurasian continent level, local central European spreading patterns suggest historic west to east spread followed by more recent east to west spread. The phylogenetic and network analyses indicate TBEV ingressions from the Czech Republic and Slovakia into Germany via landscape features (Danube river system), biogenic factors (birds, red deer) and anthropogenic factors. The identification of endemic foci showing local genetic diversity is of paramount importance to the field as these will be a prerequisite for in-depth analysis of focal TBEV maintenance and long-distance TBEV spread.

Clarifying Bunyamwera virus riddles of the past.

Pyrosequencing data and phylogenetic analysis for the full genome of Ilesha virus, Ngari virus and Calovo virus are described clarifying their much discussed relationship within the species Bunyamwera virus of the genus Orthobunyavirus of the Bunyaviridae.