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.

Establishment of a minigenome system for Oropouche virus reveals the S genome segment to be significantly longer than reported previously.

Oropouche virus (OROV) is a medically important orthobunyavirus, which causes frequent outbreaks of a febrile illness in the northern parts of Brazil. However, despite being the cause of an estimated half a million human infections since its first isolation in Trinidad in 1955, details of the molecular biology of this tripartite, negative-sense RNA virus remain limited. We have determined the complete nucleotide sequence of the Brazilian prototype strain of OROV, BeAn 19991, and found a number of differences compared with sequences in the database. Most notable were that the S segment contained an additional 204 nt at the 3' end and that there was a critical nucleotide mismatch at position 9 within the base-paired terminal panhandle structure of each genome segment. In addition, we obtained the complete sequence of the Trinidadian prototype strain TRVL-9760 that showed similar characteristics to the BeAn 19991 strain. By using a T7 RNA polymerase-driven minigenome system, we demonstrated that cDNA clones of the BeAn 19991 L and S segments expressed functional proteins, and also that the newly determined terminal untranslated sequences acted as functional promoters in the minigenome assay. By co-transfecting a cDNA to the viral glycoproteins, virus-like particles were generated that packaged a minigenome and were capable of infecting naive cells.

Detection of Klebsiella pneumoniae Carbapenem Resistance Genes by qPCR: Choosing the Right Method for Total DNA Extraction.

Rapid and accurate detection of Klebsiella pneumoniae carbapenem resistance is important for infection control and targeted antibiotic therapy. PCR-based assay performance heavily depends on the quality and quantity of template DNA. Challenges arise from the necessity to isolate chromosomal and large plasmid-encoded resistance genes simultaneously from a limited number of target cells and to remove PCR inhibitors. qPCRs for the detection of K. pneumoniae strains carrying blaOXA-48, blaNDM-1, blaKPC-2, and blaVIM-1 carbapenemase genes were developed. We compared the performance of template DNA extracted with silica column-based methods, reversed elution systems, and lysis-only methods either from diluted culture fluid or from a synthetic stool matrix which contained PCR inhibitors typically present in stool. The synthetic stool matrix was chosen to mimic K. pneumoniae containing rectal swabs or stool samples in a reproducible manner. For total DNA isolated from culture fluid, resistance gene detection by qPCR was always possible, independent of the extraction method. However, when total DNA was isolated from synthetic stool matrix spiked with K. pneumoniae, most methods were insufficient. The best performance of template DNA was obtained with reversed elution. This highlights the importance of choosing the right DNA extraction method for consistent carbapenem resistance detection by PCR.

Noninvasive indocyanine green plasma disappearance rate predicts early complications, graft failure or death after liver transplantation.

BACKGROUND: Early detection of graft malfunction or postoperative complications is essential to save patients and organs after orthotopic liver transplantation (OLT). Predictive tests for graft dysfunction are needed to enable earlier implementation of organ-saving interventions following transplantation. This study was undertaken to assess the value of indocyanine green plasma disappearance rates (ICG-PDRs) for predicting postoperative complications, graft dysfunction, and patient survival following OLT. METHODS: Eighty-six patients undergoing OLT were included in this single-centre trial. ICG-PDR was assessed daily for the first 7 days following OLT. Endpoints were graft loss or death within 30 days and postoperative complications, graft loss, or death within 30 days. RESULTS: Postoperative complications of 31 patients included deaths (12 patients) or graft losses. ICG-PDR was significantly different in patients whose endpoints were graft loss or death beginning from day 3 and in those whose endpoints were graft-loss, death, or postoperative complications beginning from day 4 after OLT. For day 7 measurements, receiver operating characteristic curve analysis revealed an ICG-PDR cut-off for predicting death or graft loss of 9.6% per min (a sensitivity of 75.0%, a specificity of 72.6%, positive predictive value 0.35, negative predictive value 0.94). For prediction of graft loss, death, or postoperative complications, the ICG-PDR cut-off was 12.3% per min (a sensitivity of 68.9%, a specificity of 66.7%, positive predictive value 0.57, negative predictive value 0.77). CONCLUSIONS: ICG-PDR measurements on postoperative day 7 are predictive of early patient outcomes following OLT. The added value over that of routinely determined laboratory parameters is low.

Severe acute respiratory syndrome coronavirus triggers apoptosis via protein kinase R but is resistant to its antiviral activity.

In this study, infection of 293/ACE2 cells with severe acute respiratory syndrome coronavirus (SARS-CoV) activated several apoptosis-associated events, namely, cleavage of caspase-3, caspase-8, and poly(ADP-ribose) polymerase 1 (PARP), and chromatin condensation and the phosphorylation and hence inactivation of the eukaryotic translation initiation factor 2alpha (eIF2alpha). In addition, two of the three cellular eIF2alpha kinases known to be virus induced, protein kinase R (PKR) and PKR-like endoplasmic reticulum kinase (PERK), were activated by SARS-CoV. The third kinase, general control nonderepressible-2 kinase (GCN2), was not activated, but late in infection the level of GCN2 protein was significantly reduced. Reverse transcription-PCR analyses revealed that the reduction of GCN2 protein was not due to decreased transcription or stability of GCN2 mRNA. The specific reduction of PKR protein expression by antisense peptide-conjugated phosphorodiamidate morpholino oligomers strongly reduced cleavage of PARP in infected cells. Surprisingly, the knockdown of PKR neither enhanced SARS-CoV replication nor abrogated SARS-CoV-induced eIF2alpha phosphorylation. Pretreatment of cells with beta interferon prior to SARS-CoV infection led to a significant decrease in PERK activation, eIF2alpha phosphorylation, and SARS-CoV replication. The various effects of beta interferon treatment were found to function independently on the expression of PKR. Our results show that SARS-CoV infection activates PKR and PERK, leading to sustained eIF2alpha phosphorylation. However, virus replication was not impaired by these events, suggesting that SARS-CoV possesses a mechanism to overcome the inhibitory effects of phosphorylated eIF2alpha on viral mRNA translation. Furthermore, our data suggest that viral activation of PKR can lead to apoptosis via a pathway that is independent of eIF2alpha phosphorylation.

Technical feasibility of 2D-3D coregistration for visualization of self-expandable microstents to facilitate coil embolization of broad-based intracranial aneurysms: an in vitro study.

INTRODUCTION: The use of self-expandable microstents for treatment of broad-based intracranial aneurysms is widely spread. However, poor fluoroscopic visibility of the stents remains disadvantageous during the coiling procedure. Flat detector angiographic computed tomography (ACT) provides high resolution imaging of microstents even though integration of this imaging modality in the neurointerventional workflow has not been widely reported. METHODS: An acrylic glass model was used to simulate the situation of a broad-based sidewall aneurysm. After insertion of a self-expandable microstent, ACT was performed. The resulting 3D dataset of the Microstent was subsequently projected into a conventional 2D fluoroscopic roadmap. This 3D visualization of the stent supported the coil embolization procedure of the in vitro aneurysm. RESULTS: In vitro 2D-3D coregistration with integration of 3D ACT data of a self-expandable microstent in a conventional 2D roadmap is feasible. CONCLUSIONS: Unsatisfying stent visibility constrains clinical cases with complex parent vessel anatomy and challenging aneurysm geometry; hence, this technique potentially may be useful in such cases. In our opinion, the clinical feasibility and utility of this new technique should be verified in a clinical aneurysm embolization study series using 2D-3D coregistration.

Inhibitors of signal peptide peptidase and subtilisin/kexin-isozyme 1 inhibit Ebola virus glycoprotein-driven cell entry by interfering with activity and cellular localization of endosomal cathepsins.

Emerging viruses such as severe fever and thrombocytopenia syndrome virus (SFTSV) and Ebola virus (EBOV) are responsible for significant morbidity and mortality. Host cell proteases that process the glycoproteins of these viruses are potential targets for antiviral intervention. The aspartyl protease signal peptide peptidase (SPP) has recently been shown to be required for processing of the glycoprotein precursor, Gn/Gc, of Bunyamwera virus and for viral infectivity. Here, we investigated whether SPP is also required for infectivity of particles bearing SFTSV-Gn/Gc. Entry driven by the EBOV glycoprotein (GP) and the Lassa virus glycoprotein (LASV-GPC) depends on the cysteine proteases cathepsin B and L (CatB/CatL) and the serine protease subtilisin/kexin-isozyme 1 (SKI-1), respectively, and was examined in parallel for control purposes. We found that inhibition of SPP and SKI-1 did not interfere with SFTSV Gn + Gc-driven entry but, unexpectedly, blocked entry mediated by EBOV-GP. The inhibition occurred at the stage of proteolytic activation and the SPP inhibitor was found to block CatL/CatB activity. In contrast, the SKI-1 inhibitor did not interfere with CatB/CatL activity but disrupted CatB localization in endo/lysosomes, the site of EBOV-GP processing. These results underline the potential of protease inhibitors for antiviral therapy but also show that previously characterized compounds might exert broader specificity than initially appreciated and might block viral entry via diverse mechanisms.

Different residues in the SARS-CoV spike protein determine cleavage and activation by the host cell protease TMPRSS2.

The spike (S) protein of severe acute respiratory syndrome coronavirus (SARS-CoV) mediates viral entry into target cells. Cleavage and activation of SARS S by a host cell protease is essential for infectious viral entry and the responsible enzymes are potential targets for antiviral intervention. The type II transmembrane serine protease TMPRSS2 cleaves and activates SARS S in cell culture and potentially also in the infected host. Here, we investigated which determinants in SARS S control cleavage and activation by TMPRSS2. We found that SARS S residue R667, a previously identified trypsin cleavage site, is also required for S protein cleavage by TMPRSS2. The cleavage fragments produced by trypsin and TMPRSS2 differed in their decoration with N-glycans, suggesting that these proteases cleave different SARS S glycoforms. Although R667 was required for SARS S cleavage by TMPRSS2, this residue was dispensable for TMPRSS2-mediated S protein activation. Conversely, residue R797, previously reported to be required for SARS S activation by trypsin, was dispensable for S protein cleavage but required for S protein activation by TMPRSS2. Collectively, these results show that different residues in SARS S control cleavage and activation by TMPRSS2, suggesting that these processes are more complex than initially appreciated.

Inhibition of cytokine gene expression and induction of chemokine genes in non-lymphatic cells infected with SARS coronavirus.

BACKGROUND: SARS coronavirus (SARS-CoV) is the etiologic agent of the severe acute respiratory syndrome. SARS-CoV mainly infects tissues of non-lymphatic origin, and the cytokine profile of those cells can determine the course of disease. Here, we investigated the cytokine response of two human non-lymphatic cell lines, Caco-2 and HEK 293, which are fully permissive for SARS-CoV. RESULTS: A comparison with established cytokine-inducing viruses revealed that SARS-CoV only weakly triggered a cytokine response. In particular, SARS-CoV did not activate significant transcription of the interferons IFN-alpha, IFN-beta, IFN-lambda1, IFN-lambda2/3, as well as of the interferon-induced antiviral genes ISG56 and MxA, the chemokine RANTES and the interleukine IL-6. Interestingly, however, SARS-CoV strongly induced the chemokines IP-10 and IL-8 in the colon carcinoma cell line Caco-2, but not in the embryonic kidney cell line 293. CONCLUSION: Our data indicate that SARS-CoV suppresses the antiviral cytokine system of non-immune cells to a large extent, thus buying time for dissemination in the host. However, synthesis of IP-10 and IL-8, which are established markers for acute-stage SARS, escapes the virus-induced silencing at least in some cell types. Therefore, the progressive infiltration of immune cells into the infected lungs observed in SARS patients could be due to the production of these chemokines by the infected tissue cells.

The Role of Phlebovirus Glycoproteins in Viral Entry, Assembly and Release.

Bunyaviruses are enveloped viruses with a tripartite RNA genome that can pose a serious threat to animal and human health. Members of the Phlebovirus genus of the family Bunyaviridae are transmitted by mosquitos and ticks to humans and include highly pathogenic agents like Rift Valley fever virus (RVFV) and severe fever with thrombocytopenia syndrome virus (SFTSV) as well as viruses that do not cause disease in humans, like Uukuniemi virus (UUKV). Phleboviruses and other bunyaviruses use their envelope proteins, Gn and Gc, for entry into target cells and for assembly of progeny particles in infected cells. Thus, binding of Gn and Gc to cell surface factors promotes viral attachment and uptake into cells and exposure to endosomal low pH induces Gc-driven fusion of the viral and the vesicle membranes. Moreover, Gn and Gc facilitate virion incorporation of the viral genome via their intracellular domains and Gn and Gc interactions allow the formation of a highly ordered glycoprotein lattice on the virion surface. Studies conducted in the last decade provided important insights into the configuration of phlebovirus Gn and Gc proteins in the viral membrane, the cellular factors used by phleboviruses for entry and the mechanisms employed by phlebovirus Gc proteins for membrane fusion. Here, we will review our knowledge on the glycoprotein biogenesis and the role of Gn and Gc proteins in the phlebovirus replication cycle.

Evidence that Processing of the Severe Fever with Thrombocytopenia Syndrome Virus Gn/Gc Polyprotein Is Critical for Viral Infectivity and Requires an Internal Gc Signal Peptide.

The severe fever with thrombocytopenia syndrome virus (SFTSV) is an emerging, highly pathogenic bunyavirus against which neither antivirals nor vaccines are available. The SFTSV glycoproteins, Gn and Gc, facilitate viral entry into host cells. Gn and Gc are generated from a precursor protein, Gn/Gc, but it is currently unknown how the precursor is converted into the single proteins and whether this process is required for viral infectivity. Employing a rhabdoviral pseudotyping system, we demonstrate that a predicted signal sequence at the N-terminus of Gc is required for Gn/Gc processing and viral infectivity while potential proprotein convertase cleavage sites in Gc are dispensable. Moreover, we show that expression of Gn or Gc alone is not sufficient for host cell entry while particles bearing both proteins are infectious, and we provide evidence that Gn facilitates Golgi transport and virion incorporation of Gc. Collectively, these results suggest that signal peptidase liberates mature Gc from the Gn/Gc precursor and that this process is essential for viral infectivity and thus constitutes a potential target for antiviral intervention.

Bifunctional chimeric SuperCD suicide gene -YCD: YUPRT fusion is highly effective in a rat hepatoma model.

AIM: To investigate the effects of catalytically superior gene-directed enzyme prodrug therapy systems on a rat hepatoma model. METHODS: To increase hepatoma cell chemosensitivity for the prodrug 5-fluorocytosine (5-FC), we generated a chimeric bifunctional SuperCD suicide gene, a fusion of the yeast cytosine deaminase (YCD) and the yeast uracil phosphoribosyltransferase (YUPRT) gene. RESULTS: In vitro stably transduced Morris rat hepatoma cells (MH) expressing the bifunctional SuperCD suicide gene (MH SuperCD) showed a clearly marked enhancement in cell killing when incubated with 5-FC as compared with MH cells stably expressing YCD solely (MH YCD) or the cytosine deaminase gene of bacterial origin (MH BCD), respectively. In vivo, MH SuperCD tumors implanted both subcutaneously as well as orthotopically into the livers of syngeneic ACI rats demonstrated significant tumor regressions (P<0.01) under both high dose as well as low dose systemic 5-FC application, whereas MH tumors without transgene expression (MH naive) showed rapid progression. For the first time, an order of in vivo suicide gene effectiveness (SuperCD>> YCD>>BCD>>>negative control) was defined as a result of a direct in vivo comparison of all three suicide genes. CONCLUSION: Bifunctional SuperCD suicide gene expression is highly effective in a rat hepatoma model, thereby significantly improving both the therapeutic index and the efficacy of hepatocellular carcinoma killing by fluorocytosine.

The antiviral effect of interferon-beta against SARS-coronavirus is not mediated by MxA protein.

Severe acute respiratory syndrome (SARS) is caused by a novel coronavirus termed SARS-CoV. No antiviral treatment has been established so far. Interferons are cytokines which induce the synthesis of several antivirally active proteins in the cell. In this study, we demonstrated that multiplication of SARS-CoV in cell culture can be strongly inhibited by pretreatment with interferon-beta. Interferon-alpha and interferon-gamma, by contrast, were less effective. The human MxA protein is one of the most prominent proteins induced by interferon-beta. Nevertheless, no interference with SARS-CoV replication was observed in Vero cells stably expressing MxA. Therefore, other interferon-induced proteins must be responsible for the strong inhibitory effect of interferon-beta against SARS-CoV.

Genetic characterization of Tribec virus and Kemerovo virus, two tick-transmitted human-pathogenic Orbiviruses.

We determined the complete genome sequences of Tribec virus (TRBV) and Kemerovo virus (KEMV), two tick-transmitted Orbiviruses that can cause diseases of the central nervous system and that are currently classified into the Great Island virus serogroup. VP2 proteins of TRBV and KEMV show very low sequence similarity to the homologous VP4 protein of tick-transmitted Great Island virus (GIV). The new sequence data support previous serological classification of these Orbiviruses into the Kemerovo serogroup, which is different from the Great Island virus serogroup. Genome segment 9 of TRBV and KEMV encodes several overlapping ORF's in the +1 reading frame relative to VP6(Hel). A co-phylogenetic analysis indicates a host switch from insect-borne Orbiviruses toward Ixodes species, which is in disagreement with previously published data.

Tetrahedral vs. polyhedral mesh size evaluation on flow velocity and wall shear stress for cerebral hemodynamic simulation.

Haemodynamic factors, in particular wall shear stresses (WSSs) may have significant impact on growth and rupture of cerebral aneurysms. Without a means to measure WSS reliably in vivo, computational fluid dynamic (CFD) simulations are frequently employed to visualise and quantify blood flow from patient-specific computational models. With increasing interest in integrating these CFD simulations into pretreatment planning, a better understanding of the validity of the calculations in respect to computation parameters such as volume element type, mesh size and mesh composition is needed. In this study, CFD results for the two most common aneurysm types (saccular and terminal) are compared for polyhedral- vs. tetrahedral-based meshes and discussed regarding future clinical applications. For this purpose, a set of models were constructed for each aneurysm with spatially varying surface and volume mesh configurations (mesh size range: 5119-258, 481 volume elements). WSS distribution on the model wall and point-based velocity measurements were compared for each configuration model. Our results indicate a benefit of polyhedral meshes in respect to convergence speed and more homogeneous WSS patterns. Computational variations of WSS values and blood velocities are between 0.84 and 6.3% from the most simple mesh (tetrahedral elements only) and the most advanced mesh design investigated (polyhedral mesh with boundary layer).

Tick-borne encephalitis virus and the immune response of the mammalian host.

Tick-borne encephalitis (TBE) is caused by Tick-borne encephalitis virus (TBEV), one of the most prevalent arboviruses in Europe and in many parts of Asia. Transmission of TBEV to humans usually occurs by bite of an infected tick or rarely by ingestion of unpasteurized milk products of infected livestock. TBEV infection induces an innate and adaptive immune response, nevertheless it is able to replicate in several cell types of the immune system at the same time which probably contributes to the spread of the virus in the human host. Furthermore, TBEV can enter the central nervous system (CNS) by yet not well understood mechanisms via the blood brain barrier (BBB) or the olfactory neurons which leads to serious neurological disorders like meningitis, encephalitis or even meningoencephalitis. In this article we review the known facts and possible hypotheses of interaction of TBEV with components of the mammalian immune system and their implications for TBEV-mediated pathogenesis.

Characterization of a sandfly fever Sicilian virus isolated during a sandfly fever epidemic in Turkey.

BACKGROUND: Phleboviruses cause sandfly fever but isolates are rare. OBJECTIVES: To analyse samples from concurrent outbreaks of suspected sandfly fever in the Mediterranean provinces of Adana, Izmir and the central province of Ankara, Turkey. STUDY DESIGN: Samples from acute cases were analysed by immunofluorescence assay (IFA). Virus isolation was attempted and pyrosequencing performed. RESULTS: In IFA 38% of 106 samples tested scored IgM positive for sandfly fever Sicillian virus (SFSV), 12% for SFSV/sandfly fever Cyprus Virus (SFCV) and only 4% for SFCV. A sandfly fever Sicilian type virus designated sandfly fever Turkey virus (SFTV) was isolated. The S-segment sequence of SFTV had a homology of 98% to that of SFCV. The M-segment sequence showed a 91.1% homology to the only SFSV sequence available. The L-segment sequence showed a homology of 58% and 60.3% to Toscana virus and Rift Valley Fever virus sequences, a partial 201nt sequence showed 95.5% homology to the SFSV Sabin strain. CONCLUSION: A new phlebovirus related to sandfly fever Sicilian virus, SFTV was isolated and characterized from acute patient material. The sandfly fever Sicilian virus activity seems to be changing in Turkey. Entomological studies are needed.

Isolation and molecular characterization of a tick-borne encephalitis virus strain from a new tick-borne encephalitis focus with severe cases in Bavaria, Germany.

Tick-borne encephalitis (TBE) is the most important viral infection transmitted by ticks in Central Europe. In Germany, where TBE was classified as a notifiable disease in 2001, a highly variable number of clinically apparent human cases was reported in the last few years, ranging from the lowest number of 238 in 2007 to a maximum of 546 in 2006. The dynamics of the virus and its vector tick remain poorly understood. We investigated a highly active TBE focus in south-eastern Germany where from 2003 to 2008 a total of 9 clinical human cases was diagnosed. Three out of these 9 cases were fatal indicating an unusually high mortality rate possibly due to a highly virulent TBEV strain. From 2005 till 2008, 2150 Ixodes ricinus ticks were collected and tested for the presence of TBE virus. Five TBEV-positive ticks were detected by real-time RT-PCR. A viable virus strain was isolated from one of the positive ticks sampled in 2005. This is the first TBE virus isolate from a tick in Germany for 30 years. Sequencing of the full-length genome of this virus strain (AS33) revealed 2 unique amino acid substitutions in the envelope protein known to play a role in the pathogenicity of TBE virus. Amplification of the envelope gene using 2 TBEV-PCR-positive ticks from 2006 also showed these particular mutations indicating that this TBE virus strain was present in at least 2 consecutive years. The entire sampling area was divided into smaller sectors for the exact location of TBEV-positive ticks. Virus-positive ticks were found to be randomly distributed throughout the investigated focus, which is used as recreational area by the local people.