HCV Defective Genomes Promote Persistent Infection by Modulating the Viral Life Cycle

Defective interfering (DI) RNAs have been detected in several human viruses. HCV in-frame deletions mutants (IFDMs), missing mainly the envelope proteins, have been found in patient sera and liver tissues. IFDMs replicate independently and can be trans-packaged into infectious virions in the presence of full length viral genome. So far, their biological role is unclear. In this study, we have isolated and cloned IFDMs from sera samples and liver tissues of patients infected with HCV genotypes 1b, 2a, and 3a. IFDMs were present in up to 26% of samples tested. Using the in vitro HCV cell culture system, co-expression of the wild type (wt) HCV replicon with HCV IFDMs RNA resulted in increased HCV replication. Additionally, co-transfection of the HCV full length genome RNA and a defective mutant missing the envelope region led to increased viral release, collectively suggesting an important biological role for IFDMs in the virus life cycle. Recently, exosomes, masters of intercellular communication, have been implicated in the transport of HCV viral genomes. We report for the first time that exosomal RNA isolated from HCV sera samples contains HCV defective genomes. We also demonstrate that inhibition of exosomal biogenesis and release influences HCV viral replication. Overall, we provide evidence that the presence of HCV IFDMs affects both viral replication and release. IFDMs exploit exosomes as means of transport, a way to evade the immune system, to spread more efficiently and possibly maintain persistent infection

SARS-CoV-2 Molecular Transmission Clusters and Containment Measures in Ten European Regions during the First Pandemic Wave

International audienceBackground: The spatiotemporal profiling of molecular transmission clusters (MTCs) using viral genomic data can effectively identify transmission networks in order to inform public health actions targeting SARS-CoV-2 spread. Methods: We used whole genome SARS-CoV-2 sequences derived from ten European regions belonging to eight countries to perform phylogenetic and phylodynamic analysis. We developed dedicated bioinformatics pipelines to identify regional MTCs and to assess demographic factors potentially associated with their formation. Results: The total number and the scale of MTCs varied from small household clusters identified in all regions, to a super-spreading event found in Uusimaa-FI. Specific age groups were more likely to belong to MTCs in different regions. The clustered sequences referring to the age groups 50–100 years old (y.o.) were increased in all regions two weeks after the establishment of the lockdown, while those referring to the age group 0–19 y.o. decreased only in those regions where schools’ closure was combined with a lockdown. Conclusions: The spatiotemporal profiling of the SARS-CoV-2 MTCs can be a useful tool to monitor the effectiveness of the interventions and to reveal cryptic transmissions that have not been identified through contact tracing

Detection of corona, HMPV, boca and rinovirus in children presenting with respiratory tract infection

Viruses are the major cause of pediatric respiratory tract infection causing significant morbidity and mortality. This study aimed to determine the distribution of human rhinovirus (hRV), human metapneumovirus (hMPV), human bocavirus (hBoV) and human coronavirus (hCoV) type 229E and OC43 in children diagnosed as having a respiratory tract infection, over the winter period of the years 2005 to 2008. We also focus on the evaluation of the genetic diversity of the newly recognized and poorly studied, within the Greek population, hMPV and hBoV positive strains, classifying them into the main, already characterized, genetic lineages for each virus and compare them with other European strains. Molecular assays used in this study included molecular methods specific for conserved regions between the different circulating strains which included a reverse - transcription PCR for the F gene of hMPV, a nested reverse - transcription PCR for the 5’-UTR of hRV, a real - time PCR for the NP-1 gene of hBoV and a reverse - transcription multiplex real - time PCR for the N gene of hCoV. These assays were employed to screen respiratory specimens, collected by clinicians of the Influenza sentinel system and of outpatient pediatric clinics, for identification of the above mentioned respiratory viruses. In total, clinical specimens from 3307 pediatric patients including males (n=1856, 56.1%) and females (n= 1451, 43.9%), aged from 0 - ≤18 yo, were enrolled in the study. In addition, nucleotide sequences were determined for representative strains of hMPV and hBoV using non-conserved regions of their viral genome.Of 3307 specimens tested, 905 (27.4%) were positive for at least one virus and included 537 rhinoviruses, 193 bocaviruses, 188 metapneumoviruses and 73 coronaviruses. Simultaneous presence of two or three viruses was observed in 86 of the above positive cases. The majority of positive cases occurred during the beginning and the end of every winter season while preschool children seemed to be infected more frequently than the older ones. Clinical symptoms more frequently associated with respiratory infection from the above mentioned viruses included cough (93%), coryza (85.6%) and fever (85%). Lower respiratory infection was observed in 31.7% of positive patients. Viral strains selected throughout the study period where further characterized at the molecular level and phylogenetic trees of the G-gene and of the VP1/VP2 gene were generated for hMPV and hBoV, respectively. Phylogenetic analysis of a total of 50 viral strains for the two viruses revealed the circulation of hMPV genetic subtype B2, presenting with a nucleotide identity of 92-100% with each other, while hBoV isolates clustered within species 1 with the majority of them (84.6%) belonging to genotype ST2. The fields of medical research and diagnosis have grown tremendously with the advent of novel molecular techniques, allowing the detection of several respiratory viruses which contributed to a high proportion of up to 27.4% to respiratory tract infections. While in Greece epidemiological study is limited to influenza virus, this kind of studies could facilitate the understanding of viral biology and pathogenesis thus contributing to an effective restriction of their transmission. The study of developmental evolution through phylogenetic analysis and genotyping characterization could further contribute to a rapid and effective response to new species that can lead to an epidemic or even pandemic with enormous socio-economic impacts.Οι λοιμώξεις του αναπνευστικού συστήματος είναι από τις πιο συνηθισμένες ασθένειες παγκοσμίως, προκαλώντας σημαντικό ποσοστό νοσηρότητας και θνησι-μότητας στους ανθρώπους. Η παρούσα διδακτορική διατριβή είχε ως σκοπό την ανάπτυξη και βελτιστοποίηση μοριακών μεθόδων για τη διερεύνηση της συμμετοχής τεσσάρων αναπνευστικών ιών, του ανθρώπινου ρινοϊού (hRV), του ανθρώπινου μεταπνευμονοϊού (hMPV), του ανθρώπινου μπόκα ιού (hBoV) και του ανθρώπινου κορόνα ιού (hCoV), τύπων 229Ε και OC43, σε λοιμώξεις του ανώτερου και κατώτε-ρου αναπνευστικού συστήματος. Ακολούθησε η μελέτη της επιδημιολογίας και η συλλογή κλινικών δεδομένων με σκοπό την πιθανή συσχέτισή τους με την εμφάνιση του κάθε ιού, καθώς και η ανάλυση της αλληλουχίας αντιπροσωπευτικών στελεχών του hMPV και του hBoV που κυκλοφορούσαν στη νότια Ελλάδα κατά τη διάρκεια της υπο-εξέτασης περιόδου.Το υλικό που χρησιμοποιήθηκε, συλλέχθηκε κατά τη χειμερινή περίοδο τριών συνεχόμενων ετών, 2005-06, 2006-07 και 2007-08, και περιελάμβανε ρινικά ή φαρυγγικά επιχρίσματα και εκπλύματα από ασθενείς ηλικίας 0-18 ετών που εμφάνιζαν συμπτώματα λοίμωξης του αναπνευστικού συστήματος. Για την ανίχνευση των παραπάνω ιών χρησιμοποιήθηκαν μοριακές μέθοδοι, με τη χρήση εκκινητών που ήταν ειδικοί για συντηρημένες περιοχές του γονιδιώματός τους, οι οποίες περιελάμβαναν μία απλή ανάστροφής μεταγραφής PCR για το γονίδιο της πρωτεΐνης σύντηξης του hMPV, μία διπλή ανάστροφής μεταγραφής PCR για την 5’ αμετάφραστη περιοχή του γονιδιώματος του hRV, μία PCR πραγματικού χρόνου για το γονίδιο της γλυκοπ-ρωτεΐνης του hBoV και μία ανάστροφής μεταγραφής πολλαπλή PCR πραγματικού χρόνου για το γονίδιο της νουκλεοκαψιδικής πρωτεΐνης του hCoV. Για την ανάλυση της αλληλουχίας των hMPV και hBoV χρησιμοποιήθηκαν μη-συντηρημένες περιοχές και η συσχέτισή τους έγινε μέσω απεικόνισης σε φυλογενετικά δέντρα.Συνολικά, 3307 δείγματα συμπεριλήφθηκαν στη μελέτη, από τα οποία 905 (27.4%) βρέθηκαν θετικά για κάποιον από τους υπό μελέτη αναπνευστικούς ιούς. Συγκεκριμένα, 537 ασθενείς βρέθηκαν θετικοί σε hRV, 193 σε hBoV, 188 σε hMPV και 73 σε hCoV. Σε 86 από τις παραπάνω περιπτώσεις παρατηρήθηκε ταυτόχρονη ανίχνευση δύο ή και τριών ιών. Το μεγαλύτερο ποσοστό ανίχνευσης παρατηρήθηκε κατά τους πρώτους και τελευταίους μήνες της υπό μελέτης περιόδου ενώ οι ασθενείς προσχολικής ηλικίας φάνηκε να είναι πιο επιρρεπείς στη λοίμωξη από τους συγκεκ-ριμένους αναπνευστικούς ιούς. Τα κλινικά συμπτώματα που εμφάνιζε η πλειοψηφία των θετικών ασθενών ήταν βήχας (93%), καταρροή (85.6%) και πυρετός (85%) ενώ υψηλά ήταν και τα ποσοστά λοίμωξης του κατώτερου αναπνευστικού συστήματος που περιελάμβανε πνευμονία και βρογχιολίτιδα. Αντιπροσωπευτικά δείγματα που βρέθηκαν θετικά για τους ιούς hMPV και hBoV κατά τις τρεις χειμερινές περιόδους επιλέχθηκαν για να μελετηθούν φυλογενετικά. Συγκεκριμένα, η σύγκριση της αλληλουχίας 24 στελεχών για τον hMPV έδειξε ότι όλα ανήκαν στη γενετική ομάδα Β2 και παρουσίαζαν μεταξύ τους νουκλεοτιδική ομολογία 91.9-100% και αμινοξική ομολογία 84.7-100%. Αντίστοιχα, η σύγκριση της αλληλου-χίας 26 στελεχών για τον hBoV έδειξε ότι η πλειοψηφία (84.6%) ανήκε στο γονότυπο St2, 2 από τα ελληνικά στελέχη ανήκαν στον γονότυπο St1, ενώ τα υπόλοιπα 2 σχημάτιζαν έναν τρίτο κλάδο που διέφερε εξίσου από τους δύο προηγούμενους γονοτύπους. Μεταξύ τους τα ελληνικά hBoV στελέχη παρουσίαζαν νουκλεοτιδική και αμινοξική ομολογία 98.2-100% και 95.6-100%, αντίστοιχα.Για την πραγματοποίηση της παρούσας διδακτορικής διατριβής καθοριστική ήταν η συμβολή των μοριακών τεχνικών καθώς η χρήση τους έχει αλλάξει ριζικά τη μελέτη του γενετικού υλικού και έχει συμβάλει σημαντικά στην ευρεία εφαρμογή της μοριακής βιολογίας στην ιατρική έρευνα και διάγνωση. Η μελέτη της επιδημιολογίας των αναπνευστικών ιών, που στην Ελλάδα περιορίζεται κυρίως στον ιό της γρίπης, μπορεί να βοηθήσει στην περαιτέρω κατανόηση της βιολογίας τους και του τρόπου παθογένειας και μετάδοσής τους ενώ η μελέτη της αναπτυξιακής εξέλιξης του γονιδιώματός τους μπορεί να συμβάλει στη γρήγορη και αποτελεσματική αντιμετώ-πιση νέων ειδών που μπορεί να οδηγήσουν σε επιδημία ή ακόμα και πανδημία με τεράστιες κοινωνικο-οικονομικές επιπτώσεις

A 5-year study of human parechoviruses in children living in bad sanitation conditions and non-polio acute flaccid paralysis children from Greece

International audienc

Targeted Virome Sequencing Enhances Unbiased Detection and Genome Assembly of Known and Emerging Viruses—The Example of SARS-CoV-2

Targeted virome enrichment and sequencing (VirCapSeq-VERT) utilizes a pool of oligos (baits) to enrich all known—up to 2015—vertebrate-infecting viruses, increasing their detection sensitivity. The hybridisation of the baits to the target sequences can be partial, thus enabling the detection and genomic reconstruction of novel pathogens with <40% genetic diversity compared to the strains used for the baits’ design. In this study, we deploy this method in multiplexed mixes of viral extracts, and we assess its performance in the unbiased detection of DNA and RNA viruses after cDNA synthesis. We further assess its efficiency in depleting various background genomic material. Finally, as a proof-of-concept, we explore the potential usage of the method for the characterization of unknown, emerging human viruses, such as SARS-CoV-2, which may not be included in the baits’ panel. We mixed positive samples of equimolar DNA/RNA viral extracts from SARS-CoV-2, coronavirus OC43, cytomegalovirus, influenza A virus H3N2, parvovirus B19, respiratory syncytial virus, adenovirus C and coxsackievirus A16. Targeted virome enrichment was performed on a dsDNA mix, followed by sequencing on the NextSeq500 (Illumina) and the portable MinION sequencer, to evaluate its usability as a point-of-care (PoC) application. Genome mapping assembly was performed using viral reference sequences. The untargeted libraries contained less than 1% of total reads mapped on most viral genomes, while RNA viruses remained undetected. In the targeted libraries, the percentage of viral-mapped reads were substantially increased, allowing full genome assembly in most cases. Targeted virome sequencing can enrich a broad range of viruses, potentially enabling the discovery of emerging viruses

Genetic variability of human metapneumo- and bocaviruses in children with respiratory tract infections

International audienceOBJECTIVES: The genotypic analysis of human metapneumo-(HMPV) and boca-(HBoV) viruses circulating in Greece and their comparison to reference and other clinical strains.DESIGN: Genetic analysis of representative strains over three consecutive winter seasons of the years 2005-2008.SETTING: Representative positive specimens for HMPV and HBoV from paediatric patients of healthcare units and hospitals in Southern Greece with influenza-like illness or other respiratory tract infections.SAMPLE: Seven to ten positive specimens for either HMPV or HBoV from each winter period. In total, 24 specimens positive for HMPV and 26 for HBoV, respectively.MAIN OUTCOME MEASURES: Sequence diversity of HMPV and HBoV strains by sequencing the complete G and VP1/VP2 genes, respectively.RESULTS: In total, 24 HMPV strains were found to have a 92-100% nucleotide and a 85.9-100% amino acid identity. Phylogenetic analysis based on the number of amino acid differences, revealed circulation of 4 different subclusters belonging to genetic lineage B2. Similarly, analysis of 26 HBoV strains indicated that 22 clustered within genotype St2, 2 into genotype St1 and the remaining 2 formed a third cluster derived from potential recombination between different St1 genotype strains. St2 HBoV genotype was observed throughout the whole observation period whereas St1 only during the second and the third winter period. Higher levels of heterogeneity were observed between HMPV compared to HBoV strains.CONCLUSIONS: Phylogenetic analysis revealed circulation of one single lineage (B2) for HMPV viruses and predominance of St2 genotype for HBoV viruses. A possible recombination between St1 genotype strains of HBoV was observed

Cocirculation of genotypes D4 and D6 in Greece during the 2005 to 2006 measles epidemic.

International audienceOne of World Health Organization's proposed methods for the establishment of measles surveillance worldwide, to achieve the elimination of measles virus by 2010, is the genetic characterization of measles wild-type virus strains. In this study, 34 measles virus strains, isolated from clinical samples during the 2005 to 2006 measles outbreak in Greece, were genotyped and studied in terms of nucleotide variation and phylogeny. Interestingly, the cocirculation of 2 different genotypes, namely, D6 and D4, was revealed. In fact, the D4 genotype has never been previously reported in Greece. Finally, although the D4 Greek strains possessed identical nucleotide sequences, the D6 isolates segregated into 3 distinct subgroups, 2 of which differed genetically and phenotypically from all GenBank deposited measles sequences. It is, thus, important to continue the epidemiologic surveillance of measles in Greece to aid future studies of measles transmission, monitor the effectiveness of measles immunization, and eventually document the elimination of the virus in our country

Laboratory investigation and phylogenetic analysis of an imported Middle East respiratory syndrome coronavirus case in Greece.

Rapid and reliable laboratory diagnosis of persons suspected of Middle East respiratory syndrome coronavirus (MERS-CoV) infection is important for timely implementation of infection control practices and disease management. In addition, monitoring molecular changes in the virus can help elucidate chains of transmission and identify mutations that might influence virus transmission efficiency. This was illustrated by a recent laboratory investigation we conducted on an imported MERS-CoV case in Greece. Two oropharyngeal swab specimens were collected on the 1st and 2nd day of patient hospitalization and tested using two real-time RT-PCR (rRT-PCR) assays targeting the UpE and Orf-1a regions of the MERS-CoV genome and RT-PCR and partial sequencing of RNA-dependent RNA polymerase and nucleocapsid genes. Serum specimens were also collected and serological test were performed. Results from the first swab sample were inconclusive while the second swab was strongly positive for MERS-CoV RNA by rRT-PCR and confirmed positive by RT-PCR and partial gene sequencing. Positive serologic test results further confirmed MERS-CoV infection. Full-length nucleocapsid and spike gene coding sequences were later obtained from the positive swab sample. Phylogenetic analysis revealed that the virus was closely related to recent human-derived MERS-CoV strains obtained in Jeddah and Makkah, Saudi Arabia, in April 2014 and dromedary camels in Saudi Arabia and Qatar. These findings were consistent with the patient's history. We also identified a unique amino acid substitution in the spike receptor binding domain that may have implications for receptor binding efficiency. Our initial inconclusive rRT-PCR results highlight the importance of collecting multiple specimens from suspect MERS-CoV cases and particularly specimens from the lower respiratory tract

HCV Defective Genomes Promote Persistent Infection by Modulating the Viral Life Cycle

Defective interfering (DI) RNAs have been detected in several human viruses. HCV in-frame deletions mutants (IFDMs), missing mainly the envelope proteins, have been found in patient sera and liver tissues. IFDMs replicate independently and can be trans-packaged into infectious virions in the presence of full length viral genome. So far, their biological role is unclear. In this study, we have isolated and cloned IFDMs from sera samples and liver tissues of patients infected with HCV genotypes 1b, 2a, and 3a. IFDMs were present in up to 26% of samples tested. Using the in vitro HCV cell culture system, co-expression of the wild type (wt) HCV replicon with HCV IFDMs RNA resulted in increased HCV replication. Additionally, co-transfection of the HCV full length genome RNA and a defective mutant missing the envelope region led to increased viral release, collectively suggesting an important biological role for IFDMs in the virus life cycle. Recently, exosomes, masters of intercellular communication, have been implicated in the transport of HCV viral genomes. We report for the first time that exosomal RNA isolated from HCV sera samples contains HCV defective genomes. We also demonstrate that inhibition of exosomal biogenesis and release influences HCV viral replication. Overall, we provide evidence that the presence of HCV IFDMs affects both viral replication and release. IFDMs exploit exosomes as means of transport, a way to evade the immune system, to spread more efficiently and possibly maintain persistent infection

Phylogenetic analyses of the MERS-CoV S and N ORFs.

<p>Midpoint-rooted phylogenetic trees of the full-length nucleocapsid (N) and spike (S) open-reading frames (ORFs) obtained from the clinical sample and published nucleotide sequences available from i) GenBank, ii) the Health Protection Agency (HPA) website (<a href="http://www.hpa.org.uk/webw/HPAweb&HPAwebStandard/HPAweb_C/1317136246479" target="_blank">http://www.hpa.org.uk/webw/HPAweb&HPAwebStandard/HPAweb_C/1317136246479</a>) and iii) the Institut Fr Virologie (IFV) website (<a href="http://www.virology-bonn.de/index.php?id=46" target="_blank">http://www.virology-bonn.de/index.php?id=46</a>). The estimated neighbor-joining trees were constructed from nucleotide alignments using MEGA version 6.06. Sequence names are derived from Genbank accession number|virus strain name|month-year of collection. Numbers in the parentheses denote additional human sequences identical to the listed sequence. An asterisk (*) denotes 2 additional identical N ORF sequences obtained from IFV, including strains MERS-CoV/Jeddah_2014_C8826 and MERS-CoV/Jeddah_2014_C9055. Two asterisks (**) denote 3 additional identical S ORF sequences obtained from IFV, including MERS-CoV/Jeddah_2014_C9055, MERS-CoV/Jeddah_2014_C7770 and MERS-CoV/Jeddah_2014_C7149. MERS-CoV sequences derived from camel specimens indicated by camel icon. Bootstrap support values (1000 replicates) 75% are plotted at the indicated internal branch nodes. Scale bars show the number of nucleotide substitutions per site.</p