Progress in human picornavirus research : New findings from the AIROPico consortium

Several research groups in Europe are active on different aspects of human picornavirus research. The AIROPico (Academia-Industry R&D Opportunities for Picornaviruses) consortium combined the disciplines of pathogenesis, diagnostics and therapy development in order to fill the gaps in our understanding of how picornaviruses cause human disease and how to combat them. AIROPico was the first EU consortium dedicated to human picornavirus research and development, and has largely accelerated and improved R&D on picornavirus biology, diagnostics and therapy. In this article, we present the progress on pathogenesis, diagnostics and treatment strategy developments for human picornaviruses resulting from the structured, translational research approach of the AIROPico consortium. We here summarize new insights in protection against infection by maternal or cross-protective antibodies, the visualisation of interactions between virus and neutralizing antibodies by cryoEM structural imaging, and the outcomes from a picornavirus-infected human 3D organoid. Progress in molecular detection and a fast typing assay for rhinovirus species are presented, as well as the identification of new compounds potentially interesting as therapeutic compounds.Peer reviewe

Coordinated Implementation of Chikungunya Virus Reverse Transcription–PCR

A preformulated chikungunya virus real-time reverse transcription–PCR, quality-confirmed oligonucleotides, and noninfectious virus controls were distributed by the European Network for the Diagnosis of Imported Viral Diseases. An international proficiency study with 31 participants demonstrated that ad hoc implementation of molecular diagnostics was feasible and successful

Rapid Molecular Detection of Zika Virus in Acute-Phase Urine Samples Using the Recombinase Polymerase Amplification Assay

Background: Currently the detection of Zika virus (ZIKV) in patient samples is done by real-time RT-PCR. Samples collected from rural area are sent to highly equipped laboratories for screening. A rapid point-ofcare test is needed to detect the virus, especially at low resource settings.  Methodology/Principal Findings: In this report, we describe the development of a reverse transcription isothermal recombinase polymerase amplification (RT-RPA) assay for the identification of ZIKV. RT-RPA assay was portable, sensitive (21 RNA molecules), and rapid (3-15 minutes). No cross-reactivity was detected to other flaviviruses, alphaviruses and arboviruses. Compared to real-time RT-PCR, the diagnostic sensitivity was 92%, while the specificity was 100%.  Conclusions/Significance: The developed assay is a promising platform for rapid point of need detection of ZIKV in low resource settings and elsewhere (e.g. during mass gathering)

A multicentre validation of Metasin: a molecular assay for the intraoperative assessment of sentinel lymph nodes from breast cancer patients

Aims: Treatment strategies for breast cancer continue to evolve. No uniformity exists in the UK for the management of node‐positive breast cancer patients. Most centres continue to use conventional histopathology of sampled sentinel lymph nodes (SLNs), which requires delayed axillary clearance in up to 25% of patients. Some use touch imprint cytology or frozen section for intraoperative testing, although both have inherent sensitivity issues. An intraoperative molecular diagnostic approach helps to overcome some of these limitations. The aim of this study was to assess the clinical effectiveness of Metasin, a molecular method for the intraoperative evaluation of SLNs. Methods and results: RNA from 3296 lymph nodes from 1836 patients undergoing SLN assessment was analysed with Metasin. Alternate slices of tissue were examined in parallel by histology. Cases deemed to be discordant were analysed by protein gel electrophoresis. There was concordance between Metasin and histology in 94.1% of cases, with a sensitivity of 92% [95% confidence interval (CI) 88–94%] and a specificity of 97% (95% CI 95–97%). Positive and negative predictive values were 88% and 98%, respectively. Over half of the discordant cases (4.4%) were ascribed to tissue allocation bias (TAB). Conclusions: Clinical validation of the Metasin assay suggests that it is sufficiently sensitive and specific to make it fit for purpose in the intraoperative setting

Gradual emergence followed by exponential spread of the SARS-CoV-2 Omicron variant in Africa.

The geographic and evolutionary origins of the SARS-CoV-2 Omicron variant (BA.1), which was first detected mid-November 2021 in Southern Africa, remain unknown. We tested 13,097 COVID-19 patients sampled between mid-2021 to early 2022 from 22 African countries for BA.1 by real-time RT-PCR. By November-December 2021, BA.1 had replaced the Delta variant in all African sub-regions following a South-North gradient, with a peak Rt of 4.1. Polymerase chain reaction and near-full genome sequencing data revealed genetically diverse Omicron ancestors already existed across Africa by August 2021. Mutations, altering viral tropism, replication and immune escape, gradually accumulated in the spike gene. Omicron ancestors were therefore present in several African countries months before Omicron dominated transmission. These data also indicate that travel bans are ineffective in the face of undetected and widespread infection

Retraction.

This is a retraction of 'Gradual emergence followed by exponential spread of the SARS-CoV-2 Omicron variant in Africa' 10.1126/science.add873

Development of one-step quantitative reverse transcription PCR for the rapid detection of flaviviruses

Background: The genus Flavivirus includes several pathogenic agents that cause severe illness in humans. Re-emergence of West Nile virus in Europe and continuous spread of certain flaviviruses such as dengue, yellow fever and Japanese encephalitis viruses represent a global danger to public health. Therefore, a rapid and accurate molecular method is required for diagnostics and epidemiological surveillance of flaviviruses. Methods: A Pan-Flavi quantitative RT-PCR assay using a Locked-Nucleic Acid probe targeting the flavivirus NS5 gene was developed and optimized to detect a wide range of flaviviruses simultaneously. The specificity and sensitivity of the Pan-Flavi assay were tested using RNA of different flaviviruses and non-flaviviruses. Furthermore, the assay was compared directly to flavivirus species-specific assays for the ability to detect flaviviruses sensitively. Results: Two degenerate primers and one Locked-Nucleic Acids probe were designed to amplify most of the flaviviruses. To increase the specificity and fluorescence signal of the Pan-Flavi assay for detection of yellow fever virus and dengue virus 4, additional primers and probes were included. Viral RNA of thirty different flaviviruses was detected, verifying the broad range specificity. The testing of this assay was successful, using standard plasmid and RNA dilutions of yellow fever virus vaccine strain, dengue virus 1 and tick-borne encephalitis virus, with a sensitivity limit of 10–100 genome copies/reaction. Also comparatively good results were achieved for detecting different flaviviruses by the Pan-Flavi assay when compared to the flavivirus species-specific assays. Conclusion: The assay is rapid, broad-range flavivirus-specific and highly sensitive making it a valuable tool for rapid detection of flaviviruses in livestock samples, epidemiological studies or as useful complement to single flavivirus-specific assays for clinical diagnosis

Association of the eNOS E298D polymorphism and the risk of myocardial infarction in the Greek population

Abstract Background Nitric oxide (NO), produced by endothelial nitric oxide synthase (eNOS), plays a key role in the regulation of vascular tone. Endothelium-derived NO exerts vasoprotective effects by suppressing platelet aggregation, leukocyte adhesion and smooth muscle cell proliferation. The E298D polymorphic variant of eNOS has been associated with myocardial infarction (MI), but data relating to this variant are divergent in Greece. Accordingly, we examined a possible association between the E298D polymorphism of the eNOS gene and MI in a subgroup of the Greek population. Methods The study population consisted of 204 patients with a history of MI and 218 control subjects. All subjects were of Greek origin and were selected from the general population of the greater Athens area. Genotyping was performed with melting curve analysis (Lightcycler system) of polymerase chain reaction amplified products using hybridization probes. Results According to the univariate findings, the risk for MI in E298D TT was 2.06 (95%CI: 1.06-4.00, p = 0.032) versus GG+GT and 2.34 (95%CI: 1.17-4.68, p = 0.016) versus GG. The risk for the T allele was estimated at 1.42 (95%CI, 1.06-1.89, p = 0.022) as compared to G allele. Regarding the additive model, one allele increase was associated with 43% higher risk of MI (OR = 1.43, 95%CI: 1.07-1.93, p = 0.018) as compared to the baseline category of homozygous GG. The positive association of TT versus GG+GT with MI risk remained even after adjusting for the main study covariates. Moreover, strong evidence was found for an increased risk for MI among carriers of the TT genotype who were smokers, hypertensive and had a family history of CAD. Conclusions This study indicates that E298D polymorphism of the eNOS gene seems to be associated with MI occurrence in the Greek population. It is possible that TT genotype is closely linked to the etiology of MI even after adjusting for known MI risk factors.</p