Sequencing and characterization of human bocavirus genomes from patients diagnosed in Southern France between 2017 and 2022.

The diversity and evolution of the genomes of human bocavirus (HBoV), which causes respiratory diseases, have been scarcely studied. Here, we aimed to obtain and characterize HBoV genomes from patients's nasopharyngeal samples collected between 2017 and 2022 period (5 years and 7 months). Next-generation sequencing (NGS) used Illumina technology after having implemented using GEMI an in-house multiplex PCR amplification strategy. Genomes were assembled and analyzed with CLC Genomics, Mafft, BioEdit, MeV, Nextclade, MEGA, and iTol. A total of 213 genomes were obtained. Phylogeny classified them all as of Bocavirus 1 (HBoV1) species. Five HBoV1 genotypic clusters determined by hierarchical clustering analysis of 27 variable genome positions were scattered over the study period although with differences in yearly prevalence. A total of 167 amino acid substitutions were detected. Besides, coinfection was observed for 52% of the samples, rhinoviruses then adenoviruses (HAdVs) being the most common viruses. Principal component analysis showed that HBoV1 genotypic cluster α tended to be correlated with HAdV co-infection. Subsequent HAdV typing for HBoV1-positive samples and negative controls demonstrated that HAdVC species predominated but HAdVB was that significantly HBoV1-associated. Overall, we described here the first HBoV1 genomes sequenced for France. HBoV1 and HAdVB association deserves further investigation.

The question of screening organ donors for hepatitis e virus: a case report of transmission by kidney transplantation in France and a review of the literature.

BACKGROUND: Hepatitis E is a potentially serious infection in organ recipients, with an estimated two-thirds of cases becoming chronic, and with a subsequent risk of cirrhosis and death. In Europe, transmission occurs most often through the consumption of raw or undercooked pork, more rarely through blood transfusion, but also after solid organ transplantation. Here we describe a case of Hepatitis E virus (HEV) infection transmitted following kidney transplantation and review the literature describing cases of HEV infection transmitted by solid organ transplantation. CASE PRESENTATION: Three weeks after kidney transplantation, the patient presented with an isolated minimal increase in GGT and hepatic cytolysis 6 months later, leading to the diagnosis of genotype 3c hepatitis E, with a plasma viral load of 6.5 log10IU/mL. In retrospect, HEV RNA was detected in the patient's serum from the onset of hepatitis, and in the donor's serum on the day of donation, with 100% identity between the viral sequences, confirming donor-derived HEV infection. Hepatitis E had a chronic course, was treated by ribavirin, and relapsed 10 months after the end of treatment. DISCUSSION: Seven cases of transmission of HEV by solid organ transplantation have been described since 2012 without systematic screening for donors, all diagnosed at the chronic infection stage; two patients died. HEV organ donor transmission may be underestimated and there is insufficient focus on immunocompromised patients in whom mild liver function test impairment is potentially related to hepatitis E. However, since HEV infection is potentially severe in these patients, and as evidence accumulates, we believe that systematic screening of organ donors should be implemented for deceased and living donors regardless of liver function abnormalities, as is already the case in the UK and Spain. In January 2024, the French regulatory agency of transplantation has implemented mandatory screening of organ donors for HEV RNA.

Sequencing of monkeypox virus from infected patients reveals viral genomes with APOBEC3-like editing, gene inactivation, and bacterial agents of skin superinfection.

A large outbreak of Monkeypox virus (MPXV) infections has arisen in May 2022 in nonendemic countries. Here, we performed DNA metagenomics using next-generation sequencing with Illumina or Nanopore technologies for clinical samples from MPXV-infected patients diagnosed between June and July 2022. Classification of the MPXV genomes and determination of their mutational patterns were performed using Nextclade. Twenty-five samples from 25 patients were studied. A MPXV genome was obtained for 18 patients, essentially from skin lesions and rectal swabbing. All 18 genomes were classified in clade IIb, lineage B.1, and we identified four B.1 sublineages (B.1.1, B.1.10, B.1.12, B.1.14). We detected a high number of mutations (range, 64-73) relatively to a 2018 Nigerian genome (genome GenBank Accession no. NC_063383.1), which were harbored by a large part of a set of 3184 MPXV genomes of lineage B.1 recovered from GenBank and Nextstrain; and we detected 35 mutations relatively to genome ON563414.3 (a B.1 lineage reference genome). Nonsynonymous mutations occurred in genes encoding central proteins, among which transcription factors and core and envelope proteins, and included two mutations that would truncate a RNA polymerase subunit and a phospholipase d-like protein, suggesting an alternative start codon and gene inactivation, respectively. A large majority (94%) of nucleotide substitutions were G > A or C > U, suggesting the action of human APOBEC3 enzymes. Finally, >1000 reads were identified as from Staphylococcus aureus and Streptococcus pyogenes for 3 and 6 samples, respectively. These findings warrant a close genomic monitoring of MPXV to get a better picture of the genetic micro-evolution and mutational patterns of this virus, and a close clinical monitoring of skin bacterial superinfection in monkeypox patients.

Observational Cohort Study of Evolving Epidemiologic, Clinical, and Virologic Features of Monkeypox in Southern France.

We enrolled 136 patients with laboratory-confirmed monkeypox during June 4-August 31, 2022, at the University Hospital Institute Méditerranée Infection in Marseille, France. The median patient age was 36 years (interquartile range 31-42 years). Of 136 patients, 125 (92%) were men who have sex with men, 15 (11%) reported previous smallpox vaccinations, and 21 (15.5%) were HIV-positive. The most frequent lesion locations were the genitals (68 patients, 53%), perianal region (65 patients, 49%), and oral/perioral area (22 patients, 17%). Lesion locations largely corresponded with the route of contamination. Most (68%) patients had isolated anal, genital, or oral lesions when they were first seen, including 56 (61%) who had >1 positive site without a visible lesion. Concurrent sexually transmitted infections were diagnosed in 19 (15%) patients, and 7 patients (5%) were asymptomatic. We recommend vaccination campaigns, intensified testing for sexually transmitted infections, and increased contact tracing to control the ongoing monkeypox outbreak.

Quasispecies Analysis of SARS-CoV-2 of 15 Different Lineages during the First Year of the Pandemic Prompts Scratching under the Surface of Consensus Genome Sequences.

The tremendous majority of SARS-CoV-2 genomic data so far neglected intra-host genetic diversity. Here, we studied SARS-CoV-2 quasispecies based on data generated by next-generation sequencing (NGS) of complete genomes. SARS-CoV-2 raw NGS data had been generated for nasopharyngeal samples collected between March 2020 and February 2021 by the Illumina technology on a MiSeq instrument, without prior PCR amplification. To analyze viral quasispecies, we designed and implemented an in-house Excel file ("QuasiS") that can characterize intra-sample nucleotide diversity along the genomes using data of the mapping of NGS reads. We compared intra-sample genetic diversity and global genetic diversity available from Nextstrain. Hierarchical clustering of all samples based on the intra-sample genetic diversity was performed and visualized with the Morpheus web application. NGS mapping data from 110 SARS-CoV-2-positive respiratory samples characterized by a mean depth of 169 NGS reads/nucleotide position and for which consensus genomes that had been obtained were classified into 15 viral lineages were analyzed. Mean intra-sample nucleotide diversity was 0.21 ± 0.65%, and 5357 positions (17.9%) exhibited significant (>4%) diversity, in ≥2 genomes for 1730 (5.8%) of them. ORF10, spike, and N genes had the highest number of positions exhibiting diversity (0.56%, 0.34%, and 0.24%, respectively). Nine hot spots of intra-sample diversity were identified in the SARS-CoV-2 NSP6, NSP12, ORF8, and N genes. Hierarchical clustering delineated a set of six genomes of different lineages characterized by 920 positions exhibiting intra-sample diversity. In addition, 118 nucleotide positions (0.4%) exhibited diversity at both intra- and inter-patient levels. Overall, the present study illustrates that the SARS-CoV-2 consensus genome sequences are only an incomplete and imperfect representation of the entire viral population infecting a patient, and that quasispecies analysis may allow deciphering more accurately the viral evolutionary pathways.

Emergence of uncommon HIV-1 non-B subtypes and circulating recombinant forms and trends in transmission of antiretroviral drug resistance in patients with primary infection during the 2013-2015 period in Marseille, Southeastern France.

Primary HIV-1 infections (PHI) with non-B subtypes are increasing in developed countries while transmission of HIV-1 harboring antiretroviral resistance-associated mutations (RAMs) remains a concern. This study assessed non-B HIV-1 subtypes and RAMs prevalence among patients with PHI in university hospitals of Marseille, Southeastern France, in 2005-2015 (11 years). HIV-1 sequences were obtained by in-house protocols from 115 patients with PHI, including 38 for the 2013-2015 period. On the basis of the phylogenetic analysis of the reverse transcriptase region, non-B subtypes were identified in 31% of these patients. They included 3 different subtypes (3A, 1C, 4F), 23 circulating recombinant forms (CRFs) (CRF02_AG, best BLAST hits being CRF 36_cpx and CRF30 in 7 and 1 cases, respectively), and 5 unclassified sequences (U). Non-B subtypes proportion increased significantly, particularly in 2011-2013 vs in 2005-2010 (P = .03). CRF02_AG viruses largely predominated in 2005-2013 whereas atypical strains more difficult to classify and undetermined recombinants emerged recently (2014-2015). The prevalence of protease, nucleos(t)ide reverse transcriptase, and first-generation nonnucleoside reverse transcriptase inhibitors-associated RAMs were 1.7% (World Health Organization [WHO] list, 2009/2.6% International AIDS Society [IAS] list, 2017), 5.2%/4.3%, and 5.2%/5.2%, respectively. Etravirine/rilpivirine-associated RAM (IAS) prevalence was 4.3%. Men who have sex with men (MSM) were more frequently infected with drug-resistant viruses than other patients (26% vs 7%; P = .011). The recent increase of these rare HIV-1 strains and the spread of drug-resistant HIV-1 among MSM in Southeastern France might be considered when implementing prevention strategies and starting therapies.

Diversity of 1,213 hepatitis C virus NS3 protease sequences from a clinical virology laboratory database in Marseille university hospitals, southeastern France.

Infection with hepatitis C virus (HCV) represents a major public health concern worldwide. Recent therapeutic advances have been considerable, HCV genotype continuing to guide therapeutic management. Since 2008, HCV genotyping in our clinical microbiology laboratory at university hospitals of Marseille, Southeastern France, has been based on NS3 protease gene population sequencing, to allow concurrent HCV genotype and protease inhibitor (PI) genotypic resistance determinations. We aimed, first, to analyze the genetic diversity of HCV NS3 protease obtained from blood samples collected between 2003 and 2013 from patients monitored at university hospitals of Marseille and detect possible atypical sequences; and, second, to identify NS3 protease amino acid patterns associated with decreased susceptibility to HCV PIs. A total of 1,213 HCV NS3 protease sequences were available in our laboratory sequence database. We implemented a strategy based on bioinformatic tools to determine whether HCV sequences are representative of our local HCV genetic diversity, or divergent. In our 2003-2012 HCV NS3 protease sequence database, we delineated 32 clusters representative of the majority HCV genetic diversity, and 61 divergent sequences. Five of these divergent sequences showed less than 85% nucleotide identity with their top GenBank hit. In addition, among the 294 sequences obtained in 2013, three were divergent relative to these 32 previously delineated clusters. Finally, we detected both natural and on-treatment genotypic resistance to HCV NS3 PIs, including a substantial prevalence of Q80K substitutions associated with decreased susceptibility to simeprevir, a second generation PI.

Hepatitis C virus of subtype 2l in Marseille, southeastern France.

The rate of eradication of chronic hepatitis C considerably increases with direct-acting antiviral agents, particularly hepatitis C virus (HCV) polymerase inhibitors. While implementing full-length HCV NS5B polymerase sequencing in our clinical microbiology laboratory, we identified atypical HCV sequences, classified as subtype 2l, from 2 patients. HCV-2l NS5B polymerase sequences were detected from 5 and 14 additional patients by screening our laboratory hepatitis virus sequence database and the NCBI GenBank sequence database. Phylogenetic analyses show unambiguously that all HCV-2l sequences are clustered apart from HCV 2 non-l sequences, which compose a second cluster. Mean (±SD) nucleotide identity between near full-length NS5B fragments of subtype 2l was 93.4 ± 0.8% (range: 92.4-95.1). Of note, all HCV-2l sequences obtained in our laboratory and in other centers were from serum samples collected in France. Analysis of the HCV-2l NS5B polymerase amino acid sequences at 30 positions critical for interaction with or resistance to HCV polymerase inhibitors showed specific patterns.

Fatal fulminant hepatitis E associated with autoimmune hepatitis and excessive paracetamol intake in Southeastern France.

We present, to our knowledge, the first case of fatal fulminant liver failure associated with hepatitis E virus infection, autoimmune hepatitis, and excessive paracetamol intake, which occurred in a 77-year-old woman. Hepatitis E testing should be performed in severe acute liver failure cases, even when another cause has been identified.

Hepatitis C virus NS3 protease genotyping and drug concentration determination during triple therapy with telaprevir or boceprevir for chronic infection with genotype 1 viruses, southeastern France.

Telaprevir and boceprevir, the two first hepatitis C virus (HCV) NS3 protease inhibitors (PIs), considerably increase rates of sustained virologic response in association with pegylated interferon and ribavirin in chronic HCV genotype 1 infections. The 30 first patients treated by telaprevir or boceprevir including anti-HCV therapies since 2011 in Marseille University hospitals, France, were monitored. HCV loads and plasmatic concentrations of telaprevir and boceprevir were determined on sequential blood samples. HCV NS3 protease gene population sequencing was performed at baseline of treatment and in case of treatment failure. Fifteen patients (including 7 co-infected with HIV) received telaprevir and the other 15 patients (including 4 co-infected with HIV) received boceprevir. At baseline, HCV NS3 protease from six patients harbored amino acid substitutions associated with PI-resistance. Treatment failure occurred at week 12 for 7 patients. Amino acid substitutions associated with PI-resistance were observed in six of these cases. HCV NS3 R155K and T54A/S mutants, all of genotype 1a, were found from four patients. Median (interquartile range) plasma concentrations were 3,092 ng/ml (2,320-3,525) for telaprevir and 486 ng/ml (265-619) for boceprevir. For HIV-HCV co-infected patients, median concentrations were 3,162 ng/ml (2,270-4,232) for telaprevir and 374 ng/ml (229-519) for boceprevir. Plasma drug concentration monitoring revealed undetectable concentrations for two patients at week 4, and probable non-adherence to therapy for another patient. These findings indicate that routine HCV NS3 protease sequencing and plasma PI concentration monitoring might be helpful to characterize cases of therapy failure, at a cost dramatically low compared to that of anti-HCV therapy.

Complete genome sequence of Tunisvirus, a new member of the proposed family Marseilleviridae.

Marseillevirus is the founding member of the proposed family Marseilleviridae, which is the second discovered family of giant viruses that infect amoebae. These viruses have been recovered from environmental water samples and, more recently, from humans. Tunisvirus was isolated from fountain water in Tunis, Tunisia, by culturing on Acanthamoeba spp. and is a new marseillevirus. We describe here its 380,011 base-pair genome. A total of 484 proteins were identified, among which 320 and 358 have an ortholog in Marseillevirus and Lausannevirus (e-value<1e-2), respectively, and 259 and 299 have best reciprocal hits with a Marseillevirus and a Lausannevirus protein, respectively. In addition, a significant hit was found in organisms other than marseilleviruses for 144 Tunisvirus proteins, indicating extensive lateral gene transfers, as has been demonstrated previously for Marseillevirus. Finally, a total of 21 ORFans were identified. Phylogeny reconstructions and analysis of the gene repertoires of marseilleviruses, including the proportion of orthologs and the mean amino acid identity between genes in pairs, suggest that the proposed family Marseilleviridae encompasses three lineages. Lineage A is composed of Marseillevirus, Cannes 8 virus and Senegalvirus; lineage B is represented by Lausannevirus alone; and lineage C has Tunisvirus as its first member. Taken together, these findings suggest that the marseilleviruses display a substantial level of diversity.

First isolation of a Marseillevirus in the Diptera Syrphidae Eristalis tenax.

OBJECTIVE: Giant viruses and amoebae are common in freshwater, where they can coexist with various insects. We screened insect larvae to detect giant viruses using a high-throughput method. METHODS: We analyzed 86 Eristalis tenax larvae obtained from stagnant water reservoirs in Tunisia. The larvae were decontaminated and then dissected to remove internal parts for coculture with Acanthamoeba polyphaga. Genome sequencing of isolated viruses was performed on a 454 Roche instrument, and comparative genomics were performed. RESULTS: One Marseillevirus, named Insectomime virus, was isolated. The genome assembly generated two scaffolds, which were 382,776 and 3,855 bp in length. Among the 477 identified predicted proteins, the best hit for 435 of the identified proteins was a Marseillevirus or Lausannevirus protein. Tunisvirus was the most closely related to Insectomime, with 446 orthologs. One Insectomime protein shared with Lausannevirus and Tunisvirus showed the highest similarity with a protein from an aphid. CONCLUSION: The isolation of a Marseillevirus from an insect expands the diversity of environments in which giant viruses have been isolated. The coexistence of larvae and giant viruses in stagnant water may explain the presence of the giant virus in the larva internal structures. This study illustrates the putative role of amoeba in lateral gene transfer not only between the organisms it phagocytoses, but also between organisms living in the same environment. © 2013 S. Karger AG, Basel.

Shan virus: a new mimivirus isolated from the stool of a Tunisian patient with pneumonia.

OBJECTIVE: Following the isolation of a Marseillevirus from the stool of a healthy young Senegalese and a Mimivirus from a Tunisian patient with pneumonia, we attempted to isolate other giant viruses of amoebae from a large human stool collection. METHODS: During the period 2010-2011, a total of 1,605 stool samples, including 115 from Tunisian patients with pneumonia, were cultured on amoebae. We used a recently developed high-throughput isolation system to detect amoebae plaque lysis on agar plates; this method allows for the testing of 100 samples per plate per week. The giant virus was identified by sequencing of genes conserved in Megavirales. RESULTS: A single giant virus, called Shan, was isolated from the stool of a Tunisian patient with pneumonia who responded poorly to antibiotics. This virus has an icosahedral shape typical of members of the family Mimiviridae and a size of 640 ± 10 nm. Phylogenetic analyses showed that Shan virus was classified as a member of Mimivirus lineage C that infects amoebae. CONCLUSION: Only one isolate was obtained in this study, suggesting that giant viruses of amoebae are rare in human stool. The isolation of Shan virus from a patient with pneumonia brings into question the etiological role of this virus and its subsequent release in stool.

Complete genome sequence of Cannes 8 virus, a new member of the proposed family "Marseilleviridae".

Marseillevirus is a giant virus that was isolated in 2007 by culturing water collected from a cooling tower in Paris, France, on Acanthamoeba polyphaga. Since then, five other marseilleviruses have been detected in environmental or human samples. The genomes of two of the six marseilleviruses have been described in detail. We describe herein the genome of Cannes 8 virus, a new member of the proposed family "Marseilleviridae." Cannes 8 virus was isolated from water collected from a cooling tower in Cannes in southeastern France. Its genome is a circular double-stranded DNA molecule with 374,041 base pairs, larger than the Marseillevirus and Lausannevirus genomes. This genome harbors 484 open reading frames predicted to encode proteins with sizes ranging from 50 to 1,537 amino acids, among which 380 (79%) and 272 (56%) are bona fide orthologs of Marseillevirus and Lausannevirus proteins, respectively. In addition, 407 and 336 predicted proteins have significant hits against Marseillevirus and Lausannevirus proteins, respectively, and 294 proteins are shared by all three marseilleviruses. The Cannes 8 virus genome has a high level of collinearity (for 96% of orthologs) with the Marseillevirus genome. About two-thirds of the Cannes 8 virus gene repertoire is composed of family ORFans. The description and annotation of the genomes of new marseilleviruses that will undoubtedly be recovered from environmental or clinical samples will be helpful to increase our knowledge of the pan-genome of the family "Marseilleviridae."

Asfarviruses and Closely Related Giant Viruses.

Acanthamoeba polyphaga mimivirus, so called because of its "mimicking microbe", was discovered in 2003 and was the founding member of the first family of giant viruses isolated from amoeba. These giant viruses, present in various environments, have opened up a previously unexplored field of virology. Since 2003, many other giant viruses have been isolated, founding new families and taxonomical groups. These include a new giant virus which was isolated in 2015, the result of the first co-culture on Vermamoeba vermiformis. This new giant virus was named "Faustovirus". Its closest known relative at that time was African Swine Fever Virus. Pacmanvirus and Kaumoebavirus were subsequently discovered, exhibiting phylogenetic clustering with the two previous viruses and forming a new group with a putative common ancestor. In this study, we aimed to summarise the main features of the members of this group of giant viruses, including Abalone Asfarvirus, African Swine Fever Virus, Faustovirus, Pacmanvirus, and Kaumoebavirus.

Simple Wastewater Preparation Protocol Applied to Monitor the Emergence of the Omicron 21L/BA.2 Variant by Genome Sequencing.

Detecting and monitoring viruses in wastewater samples have been reported as useful ways of tracking SARS-CoV-2 epidemic trends. However, there is currently no unanimously recognised method of processing samples to identify and quantify SARS-CoV-2 variants in wastewater. We aimed to implement a method that was as simple as possible in order to be used universally. In a study performed between January 2022 and June 2022 in the city of Marseille, France, we first evaluated the impact of the sample preservation strategy. We then compared ultracentrifugation to ultrafiltration and several steps of filtration to determine the optimal approach for virus concentration. As a proof-of-concept, the definitive protocol was applied to next-generation sequencing of SARS-CoV-2 in wastewater to monitor the emergence of the Omicron variant in the city. For sewage water to be processed in the week following the sampling, storage at +4 °C is sufficient, with less than 1 Ct loss. Filtration with a 5 µm syringe filter, then with a 0.8 µm filtration unit, followed by ultrafiltration was the optimal protocol, leading to an average increase of 3.24 Ct when the starting Ct was on average 38 in the wastewater. This made it possible to observe the emergence of the Omicron 21L/BA.2 variant after Omicron 21K/BA.1 by genome sequencing over a period ranging from 20 February to 10 April 2022 in agreement with observations based on patient data. To conclude, by using a simple method requiring only basic filters and a centrifuge as equipment, it is possible to accurately track the relative incidence rates and the emergence of SARS-CoV-2 variants based on sewage samples.