HPV genotyping in clinical samples using long-read single-molecule real-time sequencing.

Human papillomavirus (HPV) genotyping is widely used, particularly in combination with high-risk (HR) HPV tests for cervical cancer screening. We developed a genotyping method using sequences of approximately 800 bp in the E6/E7 region obtained by PacBio single molecule real-time sequencing (SMRT) and evaluated its performance against MY09-11 L1 sequencing and after the APTIMA HPV genotyping assay. The levels of concordance of PacBio E6/E7 SMRT sequencing with MY09-11 L1 sequencing and APTIMA HPV genotyping were 100% and 90.8%, respectively. The sensitivity of PacBio E6/EA7 SMRT was slightly greater than that of L1 sequencing and, as expected, lower than that of HR-HPV tests. In the context of cervical cancer screening, PacBio E6/E7 SMRT is then best used after a positive HPV test. PacBio E6/E7 SMRT genotyping is an attractive alternative for HR and LR-HPV genotyping of clinical samples. PacBio SMRT sequencing provides unbiased genotyping and can detect multiple HPV infections and haplotypes within a genotype.

Characterization of virus‒host recombinant variants of the hepatitis E virus.

Hepatitis E virus is a single-strand, positive-sense RNA virus that can lead to chronic infection in immunocompromised patients. Virus-host recombinant variants (VHRVs) have been described in such patients. These variants integrate part of human genes into the polyproline-rich region that could introduce new post-translational modifications (PTMs), such as ubiquitination. The aim of this study was to characterize the replication capacity of different VHRVs, namely, RNF19A, ZNF787, KIF1B, EEF1A1, RNA18, RPS17, and RPL6. We used a plasmid encoding the Kernow strain, in which the fragment encoding the S17 insertion was deleted (Kernow p6 delS17) or replaced by fragments encoding the different insertions. The HEV RNA concentrations in the supernatants and the HepG2/C3A cell lysates were determined via RT-qPCR. The capsid protein ORF2 was immunostained. The effect of ribavirin was also assessed. The HEV RNA concentrations in the supernatants and the cell lysates were higher for the variants harboring the RNF19A, ZNF787, KIF1B, RPS17, and EEF1A1 insertions than for the Kernow p6 del S17, while it was not with RNA18 or RPL6 fragments. The number of ORF2 foci was higher for RNF19A, ZNF787, KIF1B, and RPS17 than for Kernow p6 del S17. VHRVs with replicative advantages were less sensitive to the antiviral effect of ribavirin. No difference in PTMs was found between VHRVs with a replicative advantage and those without. In conclusion, our study showed that insertions did not systematically confer a replicative advantage in vitro. Further studies are needed to determine the mechanisms underlying the differences in replicative capacity. IMPORTANCE: Hepatitis E virus (HEV) is a major cause of viral hepatitis. HEV can lead to chronic infection in immunocompromised patients. Ribavirin treatment is currently used to treat such chronic infections. Recently, seven virus-host recombinant viruses were characterized in immunocompromised patients. These viruses have incorporated a portion of a human gene fragment into their genome. We studied the consequences of these insertions on the replication capacity. We found that these inserted fragments could enhance virus replication for five of the seven recombinant variants. We also showed that the recombinant variants with replicative advantages were less sensitive to ribavirin in vitro. Finally, we found that the mechanisms leading to such a replicative advantage do not seem to rely on the post-translational modifications introduced by the human gene fragment that could have modified the function of the viral protein. The mechanisms involved in improving the replication of such recombinant viruses remain to be explored.

Higher convergence of human-great ape enteric eukaryotic viromes in central African forest than in a European zoo: a One Health analysis.

Human-animal pathogenic transmissions threaten both human and animal health, and the processes catalyzing zoonotic spillover and spillback are complex. Prior field studies offer partial insight into these processes but overlook animal ecologies and human perceptions and practices facilitating human-animal contact. Conducted in Cameroon and a European zoo, this integrative study elucidates these processes, incorporating metagenomic, historical, anthropological and great ape ecological analyses, and real-time evaluation of human-great ape contact types and frequencies. We find more enteric eukaryotic virome sharing between Cameroonian humans and great apes than in the zoo, virome convergence between Cameroonian humans and gorillas, and adenovirus and enterovirus taxa as most frequently shared between Cameroonian humans and great apes. Together with physical contact from hunting, meat handling and fecal exposure, overlapping human cultivation and gorilla pillaging in forest gardens help explain these findings. Our multidisciplinary study identifies environmental co-use as a complementary mechanism for viral sharing.

SARS-CoV-2 Co-Infections and Recombinations Identified by Long-Read Single-Molecule Real-Time Sequencing.

Co-infection with at least 2 strains of virus is the prerequisite for recombination, one of the means of genetic diversification. Little is known about the prevalence of these events in SARS-CoV-2, partly because it is difficult to detect them. We used long-read PacBio single-molecule real-time (SMRT) sequencing technology to sequence whole genomes and targeted regions for haplotyping. We identified 17 co-infections with SARS-CoV-2 strains belonging to different clades in 6829 samples sequenced between January and October, 2022 (prevalence 0.25%). There were 3 Delta/Omicron co-infections and 14 Omicron/Omicron co-infections (4 cases of 21K/21L, 1 case of 21L/22A, 2 cases of 21L/22B, 4 cases of 22A/22B, 2 cases of 22B/22C and 1 case of 22B/22E). Four of these patients (24%) also harbored recombinant minor haplotypes, including one with a recombinant virus that was selected in the viral quasispecies over the course of his chronic infection. While co-infections remain rare among SARS-CoV-2-infected individuals, long-read SMRT sequencing is a useful tool for detecting them as well as recombinant events, providing the basis for assessing their clinical impact, and a precise indicator of epidemic evolution. IMPORTANCE SARS-CoV-2 variants have been responsible for the successive waves of infection over the 3 years of pandemic. While co-infection followed by recombination is one driver of virus evolution, there have been few reports of co-infections, mainly between Delta and Omicron variants or between the first 2 Omicron variants 21K_BA.1 and 21L_BA.2. The 17 co-infections we detected during 2022 included cases with the recent clades of Omicron 22A, 22B, 22C, and 22E; 24% harbored recombinant variants. This study shows that long-read SMRT sequencing is well suited to SARS-CoV-2 genomic surveillance.

Whole-genome single molecule real-time sequencing of SARS-CoV-2 Omicron.

New variants and genetic mutations of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genome can only be identified using accurate sequencing methods. Single molecule real-time (SMRT) sequencing has been used to characterize Alpha and Delta variants, but not Omicron variants harboring numerous mutations in the SARS-CoV-2 genome. This study assesses the performance of a target capture SMRT sequencing protocol for whole genome sequencing (WGS) of SARS-CoV-2 Omicron variants and compared it to that of an amplicon SMRT sequencing protocol optimized for Omicron variants. The failure rate of the target capture protocol (6%) was lower than that of the amplicon protocol (34%, p < 0.001) on our data set, and the median genome coverage with the target capture protocol (98.6% [interquartile range (IQR): 86-99.4]) was greater than that with the amplicon protocol (76.6% [IQR: 66-89.6], [p < 0.001]). The percentages of samples with >95% whole genome coverage were 64% with the target capture protocol and 19% with the amplicon protocol (p < 0.05). The clades of 96 samples determined with both protocols were 93% concordant and the lineages of 59 samples were 100% concordant. Thus, target capture SMRT sequencing appears to be an efficient method for WGS, genotyping and detecting mutations of SARS-CoV-2 Omicron variants.

Influence of Nasopharyngeal Viral Load on the Spread of the Omicron BA.2 Variant.

We used variant typing polymerase chain reaction to describe the evolution of severe acute respiratory syndrome coronavirus 2 Omicron sublineages between December 2021 and mid-March 2022. The selective advantage of the BA.2 variant over BA.1 is not due to greater nasopharyngeal viral loads.

HIV-1 resistance genotyping by ultra-deep sequencing and 6-month virological response to first-line treatment.

OBJECTIVES: To evaluate the routine use of the Sentosa ultra-deep sequencing (UDS) system for HIV-1 polymerase resistance genotyping in treatment-naïve individuals and to analyse the virological response (VR) to first-line antiretroviral treatment. METHODS: HIV drug resistance was determined on 237 consecutive samples from treatment-naïve individuals using the Sentosa UDS platform with two mutation detection thresholds (3% and 20%). VR was defined as a plasma HIV-1 virus load <50 copies/mL after 6 months of treatment. RESULTS: Resistance to at least one antiretroviral drug with a mutation threshold of 3% was identified in 29% and 16% of samples according to ANRS and Stanford algorithms, respectively. The ANRS algorithm also revealed reduced susceptibility to at least one protease inhibitor (PI) in 14.3% of samples, to one reverse transcriptase inhibitor in 12.7%, and to one integrase inhibitor (INSTI) in 5.1%. For a mutation threshold of 20%, resistance was identified in 24% and 13% of samples according to ANRS and Stanford algorithms, respectively. The 6 months VR was 87% and was similar in the 58% of patients given INSTI-based treatment, in the 16% given PI-based treatment and in the 9% given NNRTI-based treatment. Multivariate analysis indicated that the VR was correlated with the baseline HIV virus load and resistance to at least one PI at both 3% and 20% mutation detection thresholds (ANRS algorithm). CONCLUSIONS: The Vela UDS platform is appropriate for determining antiretroviral resistance in patients on a first-line antiretroviral treatment. Further studies are needed on the use of UDS for therapeutic management.

Whole-genome sequencing of SARS-CoV-2: Comparison of target capture and amplicon single molecule real-time sequencing protocols.

Fast, accurate sequencing methods are needed to identify new variants and genetic mutations of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genome. Single-molecule real-time (SMRT) Pacific Biosciences (PacBio) provides long, highly accurate sequences by circular consensus reads. This study compares the performance of a target capture SMRT PacBio protocol for whole-genome sequencing (WGS) of SARS-CoV-2 to that of an amplicon PacBio SMRT sequencing protocol. The median genome coverage was higher (p < 0.05) with the target capture protocol (99.3% [interquartile range, IQR: 96.3-99.5]) than with the amplicon protocol (99.3% [IQR: 69.9-99.3]). The clades of 65 samples determined with both protocols were 100% concordant. After adjusting for Ct values, S gene coverage was higher with the target capture protocol than with the amplicon protocol. After stratification on Ct values, higher S gene coverage with the target capture protocol was observed only for samples with Ct > 17 (p < 0.01). PacBio SMRT sequencing protocols appear to be suitable for WGS, genotyping, and detecting mutations of SARS-CoV-2.

Impact of Casirivimab-Imdevimab on Severe Acute Respiratory Syndrome Coronavirus 2 Delta Variant Nasopharyngeal Virus Load and Spike Quasispecies.

Background: The increasing use of monoclonal antibodies (mAbs) to treat coronavirus disease 2019 raises questions about their impact on the emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) mAb-resistant variants. We assessed the impact of Casirivimab-Imdevimab on SARS-CoV-2 mutations associated with reduced mAb activity in treated patients. Methods: We measured the nasopharyngeal (NP) viral load and sequenced the haplotypes of spike gene of 50 patients infected with the SARS-CoV-2 delta variant and treated with Casirivimab-Imdevimab using single-molecule real-time sequencing. Results: The NP SARS-CoV-2 viral load of patients treated with Casirivimab-Imdevimab decreased from 8.13 (interquartile range [IQR], 7.06-8.59) log10 copies/mL pretreatment to 3.67 (IQR, 3.07-5.15) log10 copies/mL 7 days later (P < .001). Of the 36 patients for whom follow-up timepoints Spike sequencing were available, none of the Spike mutations that reduced mAb activity were detected. Conclusions: Casirivimab-Imdevimab is an effective treatment for patients infected with the SARS-CoV-2 delta variant. Despite selective pressure on SARS-CoV-2 Spike quasispecies, we detected no key mutations that reduced mAb activity in our patients.

The 16S rRNA lung microbiome in mechanically ventilated patients: a methodological study.

PURPOSE: Characterization of the respiratory tract bacterial microbiome is in its infancy when compared to the gut microbiota. To limit bias mandates a robust methodology. Specific amplification of the hypervariable (V) region of the 16SrRNA gene is a crucial step. Differences in accuracy exist for one V region to another depending on the sampled environment. We aimed to assess the impact of the primer sequences targeting the V4 region currently used for gut microbiota studies in respiratory samples. Materials and methods: The original 515 F-806R primer pair targets the V4 region of the 16SrRNA gene. We compared two different 515 F-806R primer pairs before Illumina 250 paired-end sequencing for bacterial microbiome analyses of respiratory samples from critically-ill ventilated patients. "S-V4" for "Stringent V4" primer pair is used in two ongoing international projects "the Integrative Human microbiome project (iHMP)" and "the Earth microbiome project (EMP)." "R-V4" for "Relaxed V4" primer pair has been modified to reduce biases against specific environmental taxa. The optimal method was determined by concordance with conventional microbiology. Results: Twenty samples from three patients who developed a ventilator-associated pneumonia (VAP) and four who did not (control ventilated patients) were sequenced. Highly different results were obtained. "S-V4" provided the best agreement with the conventional microbiology for endotracheal aspirate: 89% as compared to 56% for "R-V4." The main difference related to poor Enterobacteriaceae detection with "R-V4" primers. Conclusions: Accuracy of the bacterial lung microbiome composition was highly dependent on the primers used for amplification of the 16 s rRNA hypervariable sequence. This work validates for future lung microbiome studies the use of the 515 F-806R "S-V4" primer pair associated to Illumina® MiSeq paired-end sequencing.

Influence of treatment with neutralizing monoclonal antibodies on the SARS-CoV-2 nasopharyngeal load and quasispecies.

OBJECTIVES: To evaluate the impact of neutralizing monoclonal antibody (mAb) treatment and to determine whether the selective pressure of mAbs could facilitate the proliferation of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants with spike protein mutations that might attenuate mAb effectiveness. PATIENTS AND METHODS: We evaluated the impact of mAbs on the nasopharyngeal (NP) viral load and virus quasispecies of mAb-treated patients using single-molecule real-time sequencing. The mAbs used were: Bamlanivimab alone (four patients), Bamlanivimab/Etesevimab (23 patients) and Casirivimab/Imdevimab (five patients). RESULTS: The NP SARS-CoV-2 viral load of mAb-treated patients decreased from 8.2 log10 copies/mL before administration to 4.3 log10 copies/mL 7 days after administration. Five immunocompromised patients given Bamlanivimab/Etesevimab were found to have mAb activity-reducing spike mutations. Two patients harboured SARS-CoV-2 variants with a Q493R spike mutation 7 days after administration, as did a third patient 14 days after administration. The fourth patient harboured a variant with a Q493K spike mutation 7 days post-treatment, and the fifth patient had a variant with a E484K spike mutation on day 21. The emergence of the spike mutation was accompanied by stabilization or rebound of the NP viral load in three of five patients. CONCLUSION: Two-mAb therapy can drive the selection of resistant SARS-CoV-2 variants in immunocompromised patients. Patients given mAbs should be closely monitored and measures to limit virus spread should be reinforced.

Prediction of SARS-CoV-2 Variant Lineages Using the S1-Encoding Region Sequence Obtained by PacBio Single-Molecule Real-Time Sequencing.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is the causal agent of the COVID-19 pandemic that emerged in late 2019. The outbreak of variants with mutations in the region encoding the spike protein S1 sub-unit that can make them more resistant to neutralizing or monoclonal antibodies is the main point of the current monitoring. This study examines the feasibility of predicting the variant lineage and monitoring the appearance of reported mutations by sequencing only the region encoding the S1 domain by Pacific Bioscience Single Molecule Real-Time sequencing (PacBio SMRT). Using the PacBio SMRT system, we successfully sequenced 186 of the 200 samples previously sequenced with the Illumina COVIDSeq (whole genome) system. PacBio SMRT detected mutations in the S1 domain that were missed by the COVIDseq system in 27/186 samples (14.5%), due to amplification failure. These missing positions included mutations that are decisive for lineage assignation, such as G142D (n = 11), N501Y (n = 6), or E484K (n = 2). The lineage of 172/186 (92.5%) samples was accurately determined by analyzing the region encoding the S1 domain with a pipeline that uses key positions in S1. Thus, the PacBio SMRT protocol is appropriate for determining virus lineages and detecting key mutations.

Influence of SARS-CoV-2 Variant B.1.1.7, Vaccination, and Public Health Measures on the Spread of SARS-CoV-2.

The spread of SARS-CoV-2 and the resulting disease COVID-19 has killed over 2.6 million people as of 18 March 2021. We have used a modified susceptible, infected, recovered (SIR) epidemiological model to predict how the spread of the virus in regions of France will vary depending on the proportions of variants and on the public health strategies adopted, including anti-COVID-19 vaccination. The proportion of SARS-CoV-2 variant B.1.1.7, which was not detected in early January, increased to become 60% of the forms of SARS-CoV-2 circulating in the Toulouse urban area at the beginning of February 2021, but there was no increase in positive nucleic acid tests. Our prediction model indicates that maintaining public health measures and accelerating vaccination are efficient strategies for the sustained control of SARS-CoV-2.

Deciphering an Adenovirus F41 Outbreak in Pediatric Hematopoietic Stem Cell Transplant Recipients by Whole-Genome Sequencing.

Human adenovirus (HAdV) represents a major cause of mortality and morbidity in pediatric recipients of allogeneic hematopoietic stem cell transplants (HSCT). HAdV species F type 41 (HAdV-F41) infections in HSCT patients are scarce, whereas HAdV-F41 circulates commonly in healthy individuals. Between March and July 2018, HAdV-F41 infections were identified in four children (A, B, C, and E) who received allogeneic HSCT and one child before HSCT (D) at Robert Debré Hospital, Paris, France. We report here the clinical course of HAdV-F41 infection and the phylogenetic investigation to identify interpatient transmission. HAdV DNA was quantified in stool and plasma samples by real-time PCR. HAdV type was determined by sequencing of the fiber and hexon genes. Phylogenetic investigation was done with whole-genome sequences obtained by next-generation sequencing. HAdV loads in stool samples ranged from 6.60 to 10.10 log10 copies/ml. HAdV-F41 detection in plasma was observed in four patients, but no disseminated disease was reported. Two patients died, but neither death was attributed to HAdV. While sequencing limited to the fiber gene suggested a cluster with four patients, phylogenetic analysis with whole-genome sequencing (WGS) and HVR7 revealed a cluster that included three patients (C, D, and E), suggesting an interpatient transmission in that cluster and two other independent infections. HAdV-F41 levels in stool specimens of pediatric HSCT patients are high and represent a risk of interpatient transmission. WGS helped to identify related cases. Prompt detection of HAdV in stool and control measures are warranted to limit any risk of nosocomial transmission.

A multi-disciplinary comparison of great ape gut microbiota in a central African forest and European zoo.

Comparisons of mammalian gut microbiota across different environmental conditions shed light on the diversity and composition of gut bacteriome and suggest consequences for human and animal health. Gut bacteriome comparisons across different environments diverge in their results, showing no generalizable patterns linking habitat and dietary degradation with bacterial diversity. The challenge in drawing general conclusions from such studies lies in the broad terms describing diverse habitats ("wild", "captive", "pristine"). We conducted 16S ribosomal RNA gene sequencing to characterize intestinal microbiota of free-ranging sympatric chimpanzees and gorillas in southeastern Cameroon and sympatric chimpanzees and gorillas in a European zoo. We conducted participant-observation and semi-structured interviews among people living near these great apes to understand better their feeding habits and habitats. Unexpectedly, bacterial diversity (ASV, Faith PD and Shannon) was higher among zoo gorillas than among those in the Cameroonian forest, but zoo and Cameroonian chimpanzees showed no difference. Phylogeny was a strong driver of species-specific microbial composition. Surprisingly, zoo gorilla microbiota more closely resembled that of zoo chimpanzees than of Cameroonian gorillas. Zoo living conditions and dietary similarities may explain these results. We encourage multidisciplinary approach integrating environmental sampling and anthropological evaluation to characterize better diverse environmental conditions of such investigations.

Classification of the Zoonotic Hepatitis E Virus Genotype 3 Into Distinct Subgenotypes.

Hepatitis E virus (HEV) genotype 3 is the most common genotype linked to HEV infections in Europe and America. Three major clades (HEV-3.1, HEV-3.2, and HEV-3.3) have been identified but the overlaps between intra-subtype and inter-subtype p-distances make subtype classification inconsistent. Reference sequences have been proposed to facilitate communication between researchers and new putative subtypes have been identified recently. We have used the full or near full-length HEV-3 genome sequences available in the Genbank database (April 2020; n = 503) and distance analyses of clades HEV-3.1 and HEV-3.2 to determine a p-distance cut-off (0.093 nt substitutions/site) in order to define subtypes. This could help to harmonize HEV-3 genotyping, facilitate molecular epidemiology studies and investigations of the biological and clinical differences between HEV-3 subtypes.