Mutagenesis of the conserved 51-nucleotide region of Sindbis virus

We have constructed 25 site-specific mutations in a domain of 51 nucleotides in Sindbis virus that is highly conserved among all alphaviruses sequenced to date. These 51 nucleotides are capable of forming two hairpin structures and are found from nucleotides 155 to 205 in Sindbis virus within the region encoding nsP1. Of the mutations, 21 were silent and did not lead to a change in the amino acid sequence encoded. These silent mutations changed not only the linear sequence but also the stability of the hairpins in most cases. Two double mutants that were constructed led to the replacement of one base pair by another so that the linear sequence was altered but the nature of the hairpins was not. All of the mutants with silent mutations were viable, but 19 of the 21 mutants were severely impaired for growth in both chicken and mosquito cells. Compared with the parental virus, they grew slowly and produced virus at rates of 10(-1) to 10(-4) times the parental rate. Surprisingly, however, the plaques produced by these mutants were indistinguishable from those produced by the parental virus. Two of the silent mutations, found within the first hairpin structure, produced virus at a faster rate than the parental virus. It is clear that the exact sequence of this region is important for some aspect of virus replication. We suggest that one or more proteins, either virus encoded or cellular, bind to the hairpin structures in a sequence-specific fashion in a step that promotes replication of the viral RNA. Of the mutations that resulted in a change of coding, only one of four was viable, suggesting that the amino acid sequence encoded in this domain is essential for virus replication

Defined mutations in the 5' nontranslated sequence of Sindbis virus RNA

We have constructed 24 deletion mutants which contain deletions of from 1 to 15 nucleotides in the 5' nontranslated region of Sindbis virus RNA and tested the effect of these mutations on virus replication. The results showed that the first 44 nucleotides, which are capable of forming a hairpin structure, are important for virus replication, as all deletions tested in this region were either lethal or resulted in virus that grew poorly in comparison to the parental virus. Many of these deletions had different effects in mosquito cells than in chicken cells, suggesting that cellular factors, presumably proteins, bind to this region. This domain may function in at least two processes in viral replication. It seems likely that in the minus strand, this sequence element is bound by the viral replicase and promotes RNA replication. In the plus strand, this element may modulate initiation of translation of the nonstructural proteins. The results suggest that the hairpin structure itself is important. All deletions within it had deleterious effects on virus replication, and in particular, deletion of one of the G residues at nucleotide 7 or 8 or of one of the C residues at nucleotide 36 or 37 which are theoretically base-paired with these G's resulted in temperature-sensitive viruses that behaved very similarly. In contrast, large deletions between the 44-nucleotide hairpin and the translation start site at nucleotides 60 to 62 resulted in virus that grew as well as or better than the parental virus in both chicken and mosquito cells. The A residue at position 5 of the HRSP strain used was examined in more detail. Deletion of this A was lethal, whereas substitution by G resulted in a virus that grew poorly, despite the fact that G is present at position 5 in the AR339 parent of HRSP. U at position 5 resulted in a virus that grew less well than the A5 strain but better than the G5 mutant

Evolution of endogenous analgesia

Endogenous pain modulation is a complex phenomenon involved in the perception of pain. It consists of top-down inhibitory and facilitatory pathways that originate at higher sites within the central nervous system and converge at dorsal horn neurons in the spinal cord, to modulate incoming afferent nociceptive information. Dysfunction of inhibitory pain pathways or a shift in the balance between pain facilitation and pain inhibition has been associated with the development of chronic pain. This thesis describes the effect of several central-acting drugs on descending control of pain in both healthy volunteers and chronic pain patients to further understand the underlying mechanism of endogenous pain control in health and disease.UBL - phd migration 201

Enterovirus Infections in Solid Organ Transplant Recipients:a Clinical Comparison from a Regional University Hospital in the Netherlands

Enterovirus infections are known to cause a diverse range of illnesses, even in healthy individuals. However, information detailing enterovirus infections and their severity in immunocompromised patients, such as transplant recipients, is limited. We compared enterovirus infections in terms of genotypes, clinical presentation, and severity between transplant and nontransplant patients. A total of 264 patients (38 transplant recipients) with 283 enterovirus infection episodes were identified in our hospital between 2014 and 2018. We explored the following factors associated with enterovirus infections: clinical presentation and diagnosis on discharge, length of hospital stay, symptom persistence, and infection episodes in both children and adults. We observed some differences in genotypes between patients, with enterovirus group C occurring mainly in transplant recipients (P < 0.05). EV-associated gastrointestinal infections were more common in patients with a transplant (children [71%] and adults [46%]), compared to nontransplant patients (P < 0.05). Additionally, nontransplant patients had a higher number of hospital stays (P < 0.05), potentially reflecting more severe disease. However, transplant patients were more likely to have symptom persistence after discharge (P < 0.05). Finally, children and adults with a transplant were more likely to have additional enterovirus infection episodes (P < 0.05). In our cohort, enterovirus infections did not seem to be more severe after transplantation; however, patients tended to present with different clinical symptoms and had genotypes rarely found in nontransplant recipients. IMPORTANCE Despite the high prevalence of enteroviruses in the community and the increasing demand for transplants from an aging population, knowledge on enteroviruses in solid organ transplant recipients is currently limited. Transplant recipients represent a significant patient population and require additional considerations in patient management, particularly as they have an increased risk of disease severity. Enteroviruses are known to cause significant morbidity, with a diverse range of clinical presentation from over 100 different genotypes. In this study, we aimed to provide a more comprehensive overview of enteroviral infections in transplant recipients, compared to nontransplant patients, and to bridge some gaps in our current knowledge. Identifying potential clinical manifestation patterns can help improve patient management following enterovirus infections

Validation and verification of the GeneFinder (TM) COVID-19 Plus RealAmp kit on the ELITe InGenius (R) instrument

Background: Throughout the SARS-CoV-2 pandemic, a rapid identification of the virus was essential to quickly recognize positive cases and limit further spread by applying appropriate infection prevention. Many diagnostic laboratories use a multiplex Real-Time PCR assay, as they are not only highly sensitive but also specific. Currently, there are several assays and platforms in the market available which target different SARS-CoV-2 genes. The aim of this study was to validate and verify the GeneFinder (TM) COVID-19 PLUS RealAmp kit on the ELITe InGenius (R) instrument and compare to the national reference method. Methods: GeneFinder (TM) COVID-19 PLUS RealAmp kit was evaluated against the routine WHO in- house RealTime PCR assay, which is also the national reference method in the Netherlands and used in our laboratory. The sensitivity was tested using the analytical panel from Qnostics (Glasgow, United Kingdom) and the specificity was tested with patient material comprising of other seasonal respiratory viruses. In addition, 96 clinical samples initially analyzed by routine Real-Time PCR were tested using the GeneFinder (TM) COVID-19 PLUS RealAmp kit on the ELITe InGenius (R) instrument. Results: The GeneFinder (TM) COVID-19 PLUS RealAmp kit had a similar performance compared to routine in-house testing, with a limit of detection of 500 dC/mL for the RdRp-gene and E gene. Meanwhile, the N gene showed a limit of detection of 50 dC/mL. The SARS-CoV-2 test was highly specific and detected no other respiratory viruses. The results of the clinical samples were comparable between both assays with similar Ct values observed for the in-house Real-Time-PCR and the GeneFinder (TM) COVID-19 PLUS RealAmp kit for the N gene. Conclusion: The GeneFinder (TM) COVID-19 PLUS RealAmp kit on the ELITe InGenius (R) instrument had an appropriate sensitivity and specificity that could be used in small scale laboratories or during night shifts where accurate diagnostics are crucial

Newly Identified Enterovirus C Genotypes, Identified in the Netherlands through Routine Sequencing of All Enteroviruses Detected in Clinical Materials from 2008 to 2015

Enteroviruses (EVs) are a group of human and animal viruses that are capable of causing a variety of clinical syndromes. Different genotypes classified into species can be distinguished on the basis of sequence divergence in the VP1 capsid-coding region. Apparently new genotypes are discovered regularly, often as incidental findings in studies investigating respiratory syndromes or as part of poliovirus surveillance. Recently, some EVs have become recognized as significant respiratory pathogens, and a number of new genotypes belonging to species C have been identified. The circulation of these newly identified species C EVs, such as EV-C104, EV-C105, EV-C109, and EV-C117, nevertheless appears to be limited. In this report, we show the results of routine genotyping of all enteroviruses detected in our tertiary care hospital between January 2008 and April 2015. We detected 365 EVs belonging to 40 genotypes. Interestingly, several newly identified species C EVs were detected during the study period. Sequencing of the 5' untranslated region (5'UTR) of these viruses shows divergence in this region, which is a target region in many detection assays

Enterovirus D68-The New Polio?

Enterovirus D68 (EV-D68) has emerged over the recent years, with large outbreaks worldwide. Increased occurrence has coincided with improved clinical awareness and surveillance of non-polio enteroviruses. Studies showing its neurotropic nature and the change in pathogenicity have established EV-D68 as a probable cause of Acute Flaccid Myelitis (AFM). The EV-D68 storyline shows many similarities with poliovirus a century ago, stimulating discussion whether EV-D68 could be ascertaining itself as the "new polio." Increasing awareness amongst clinicians, incorporating proper diagnostics and integrating EV-D68 into accessible surveillance systems in a way that promotes data sharing, will be essential to reveal the burden of disease. This will be a necessary step in preventing EV-D68 from becoming a threat to public health

A discussion of syndromic molecular testing for clinical care

Current molecular detection methods for single or multiplex pathogens by real-time PCR generally offer great sensitivity and specificity. However, many infectious pathogens often result in very similar clinical presentations, complicating the test-order for physicians who have to narrow down the causative agent prior to in-house PCR testing. As a consequence, the intuitive response is to start empirical therapy to treat a broad spectrum of possible pathogens. Syndromic molecular testing has been increasingly integrated into routine clinical care, either to provide diagnostic, epidemiological or patient management information. These multiplex panels can be used to screen for predefined infectious disease pathogens simultaneously within a 1 h timeframe, creating opportunities for rapid diagnostics. Conversely, syndromic panels have their own challenges and must be adaptable to the evolving demands of the clinical setting. Firstly, questions have been raised regarding the clinical relevance of some of the targets included in the panels and secondly, there is the added expense of integration into the clinical laboratory. Here, we aim to discuss some of the factors that should be considered before performing syndromic testing rather than traditional low-plex in-house PCR