Alternative splicing of viral transcripts: the dark side of HBV.

Regulation of alternative splicing is one of the most efficient mechanisms to enlarge the proteomic diversity in eukaryotic organisms. Many viruses hijack the splicing machinery following infection to accomplish their replication cycle. Regarding the HBV, numerous reports have described alternative splicing events of the long viral transcript (pregenomic RNA), which also acts as a template for viral genome replication. Alternative splicing of HBV pregenomic RNAs allows the synthesis of at least 20 spliced variants. In addition, almost all these spliced forms give rise to defective particles, detected in the blood of infected patients. HBV-spliced RNAs have long been unconsidered, probably due to their uneasy detection in comparison to unspliced forms as well as for their dispensable role during viral replication. However, recent data highlighted the relevance of these HBV-spliced variants through (1) the trans-regulation of the alternative splicing of viral transcripts along the course of liver disease; (2) the ability to generate defective particle formation, putative biomarker of the liver disease progression; (3) modulation of viral replication; and (4) their intrinsic propensity to encode for novel viral proteins involved in liver pathogenesis and immune response. Altogether, tricky regulation of HBV alternative splicing may contribute to modulate multiple viral and cellular processes all along the course of HBV-related liver disease.

Multisystem inflammatory syndrome in children rose and fell with the first wave of the COVID-19 pandemic in France.

AIM: This study determined the influence of the COVID-19 pandemic on the occurrence of multisystem inflammatory syndrome in children (MIS-C) and compared the main characteristics of MIS-C and Kawasaki disease (KD). METHODS: We included patients aged up to 18 years of age who were diagnosed with MIS-C or KD in a paediatric university hospital in Paris from 1 January 2018 to 15 July 2020. Clinical, laboratory and imaging characteristics were compared, and new French COVID-19 cases were correlated with MIS-C cases in our hospital. RESULTS: There were seven children with MIS-C, from 6 months to 12 years of age, who were all positive for the virus that causes COVID-19, and 40 virus-negative children with KD. Their respective characteristics were as follows: under 5 years of age (14.3% vs. 85.0%), paediatric intensive care unit admission (100% vs. 10.0%), abdominal pain (71.4% vs. 12.5%), myocardial dysfunction (85.7% vs. 5.0%), shock syndrome (85.7% vs. 2.5%) and mean and standard deviation C-reactive protein (339 ± 131 vs. 153 ± 87). There was a strong lagged correlation between the rise and fall in MIS-C patients and COVID-19 cases. CONCLUSION: The rise and fall of COVID-19 first wave mirrored the MIS-C cases. There were important differences between MIS-C and KD.

Cardiac MRI in Children with Multisystem Inflammatory Syndrome Associated with COVID-19.

This case series examines cardiac MRI findings in four children and adolescents admitted to intensive care in April 2020 for multisystem inflammatory syndrome and Kawasaki disease-like features related to coronavirus disease 2019 (COVID-19). Acute myocarditis occurred less than 1 week after onset of fever and gastrointestinal symptoms. Physical examination showed rash and cheilitis or conjunctivitis. All patients recovered after intravenous immunoglobulin therapy. Severe acute respiratory syndrome coronavirus 2 reverse transcription polymerase chain reaction was negative in nasopharyngeal, stool, and respiratory samples and was positive on serology. Cardiac MRI showed diffuse myocardial edema on T2 short tau inversion-recovery sequences and native T1 mapping, with no evidence of late gadolinium enhancement suggestive of replacement fibrosis or focal necrosis. These findings favor postinfectious myocarditis in children and adolescents with COVID-19.

Fetal Brain Injury Associated with Parvovirus B19 Congenital Infection Requiring Intrauterine Transfusion.

BACKGROUND: Infection with parvovirus B19 (B19V) during pregnancy may cause severe fetal anemia, hydrops, and fe tal death. Furthermore, neurodevelopmental impairment among survivors may occur despite appropriate prenatal management, including intrauterine transfusion (IUT). OBJECTIVES: Our primary objective was to describe cerebral lesions on MRI in fetuses with severe anemia requiring IUT for B19V infection. Our secondary objective was to search for clinical and biological characteristics associated with the occurrence of such lesions. STUDY DESIGN: We performed a retrospective review of data on fetuses infected with B19V and requiring at least one IUT between 2005 and 2016. Fetuses with abnormal cerebral MRI results in the 3rd trimester were compared to those with normal MRI results. RESULTS: Of 34 transfused fetuses, 26 children were born at full term. Five intrauterine fetal deaths, 1 neonatal death, and 2 terminations of pregnancy occurred. Cerebral anomalies were observed in 7/27 fetuses on MRI, including cerebellar hemorrhage or a small cerebellum. Only viral load in fetal blood appeared to be associated with brain lesions (11.5 log10 copies/mL [10.5-12.5] in case of abnormal MRI results vs. 9.5 log10 copies/mL [7.8-10.0]; p = 0.05). CONCLUSIONS: Among the fetuses transfused for B19V infection, 26% presented with prenatal abnormal cerebral imaging results. In our study, viral load in fetal blood appeared to be the only factor associated with fetal brain lesions.

Alternative splicing of hepatitis B virus: A novel virus/host interaction altering liver immunity.

BACKGROUND & AIMS: Hepatitis B virus (HBV) RNA can undergo alternative splicing, but the relevance of this post-transcriptional regulation remains elusive. The mechanism of HBV alternative splicing regulation and its impact on liver pathogenesis were investigated. METHODS: HBV RNA-interacting proteins were identified by RNA pull-down, combined with mass spectrometry analysis. HBV splicing regulation was investigated in chemically and surgically induced liver damage, in whole HBV genome transgenic mice and in hepatoma cells. Viral and endogenous gene expression were quantified by quantitative reverse transcription polymerase chain reaction, Western blot and enzyme-linked immunosorbent assay. Resident liver immune cells were studied by fluorescence-activated cell sorting. RESULTS: HBV pregenomic RNA-interacting proteins were identified and 15% were directly related to the splicing machinery. Expression of these splicing factors was modulated in HBV transgenic mice with liver injuries and contributed to an increase of the HBV spliced RNA encoding for HBV splicing-generated protein (HBSP). HBSP transgenic mice with chemically induced liver fibrosis exhibited attenuated hepatic damage. The protective effect of HBSP resulted from a decrease of inflammatory monocyte/macrophage recruitment through downregulation of C-C motif chemokine ligand 2 (CCL2) expression in hepatocytes. In human hepatoma cells, the ability of HBSP to control CCL2 expression was confirmed and maintained in a whole HBV context. Finally, viral spliced RNA detection related to a decrease of CCL2 expression in the livers of HBV chronic carriers underscored this mechanism. CONCLUSION: The microenvironment, modified by liver injury, increased HBSP RNA expression through splicing factor regulation, which in turn controlled hepatocyte chemokine synthesis. This feedback mechanism provides a novel insight into liver immunopathogenesis during HBV infection. Lay summary: Hepatitis B virus persists for decades in the liver of chronically infected patients. Immune escape is one of the main mechanisms developed by this virus to survive. Our study highlights how the crosstalk between virus and liver infected cells may contribute to this immune escape.

Transfection of exogenous rotavirus rearranged RNA segments in cells infected with a WT rotavirus results in subsequent gene rearrangements.

Group A rotaviruses, members of the family Reoviridae, are a major cause of infantile acute gastroenteritis. The rotavirus genome consists of 11 dsRNA segments. In some cases, an RNA segment is replaced by a rearranged RNA segment, which is derived from its standard counterpart by partial sequence duplication. It has been shown that some rearranged segments are preferentially encapsidated into viral progenies after serial passages in cell culture. Based on this characteristic, a reverse genetics system was used previously to introduce exogenous segment 7 rearrangements into an infectious rotavirus. This study extends this reverse genetics system to RNA segments 5 and 11. Transfection of exogenous rotavirus rearranged RNA segment 5 or 11 into cells infected with a WT helper rotavirus (bovine strain RF) resulted in subsequent gene rearrangements in the viral progeny. Whilst recombinant viruses were rescued with an exogenous rearranged segment 11, the exogenous segment was modified by a secondary rearrangement. The occurrence of spontaneous rearrangements of WT or exogenous segments is a major hindrance to the use of this reverse genetics approach.

Superior control of HIV-1 replication by CD8+ T cells is reflected by their avidity, polyfunctionality, and clonal turnover.

The key attributes of CD8+ T cell protective immunity in human immunodeficiency virus (HIV) infection remain unclear. We report that CD8+ T cell responses specific for Gag and, in particular, the immunodominant p24 epitope KK10 correlate with control of HIV-1 replication in human histocompatibility leukocyte antigen (HLA)-B27 patients. To understand further the nature of CD8+ T cell-mediated antiviral efficacy, we performed a comprehensive study of CD8+ T cells specific for the HLA-B27-restricted epitope KK10 in chronic HIV-1 infection based on the use of multiparametric flow cytometry together with molecular clonotypic analysis and viral sequencing. We show that B27-KK10-specific CD8+ T cells are characterized by polyfunctional capabilities, increased clonal turnover, and superior functional avidity. Such attributes are interlinked and constitute the basis for effective control of HIV-1 replication. These data on the features of effective CD8+ T cells in HIV infection may aid in the development of successful T cell vaccines.

Rapid molecular diagnosis of measles virus infection in an epidemic setting.

During the 2011 measles outbreak in Paris (France), patients with clinical suspicion of measles were tested for virological confirmation of measles virus (MV) infection. To assess the practical value of molecular diagnosis in an epidemic setting, 171 oral fluid samples and 235 serum samples collected from 270 patients were tested prospectively for MV-RNA using a novel one-step real-time RT-PCR assay including an internal control. Serum samples were also tested for MV-specific IgG and IgM antibodies. MV infection was confirmed by detection of MV-RNA and/or MV-IgM for 229 of the 270 patients. The results for the 102 cases with both serum and oral fluid samples available were used to compare the techniques. The detection rate of MV-RNA by RT-PCR was 98% (100/102) for oral fluid and 95% (97/102) for serum samples. The detection rate of MV-IgM was 85% (87/102). Negative MV-IgM results were observed mostly for serum samples collected early after the onset of the rash. A MV-RNA standard of known concentration obtained by in vitro transcription was used to quantify MV-RNA in samples. MV-RNA copy numbers were significantly higher in oral fluid than in serum samples, but did not correlate with time of sampling (within 1 week after the onset of the rash), patient age, or vaccination status. During the early stage of infection, the MV-RNA viral load in serum was lower in patients positive than in those negative for MV-IgG. In conclusion, the one-step real-time RT-PCR assay is a simple and sensitive tool suitable for MV diagnosis within hours.

Differential Performance of the FilmArray Meningitis/Encephalitis Assay To Detect Bacterial and Viral Pathogens in Both Pediatric and Adult Populations.

Meningitis/encephalitis (ME) syndromic diagnostic assays can be applied for the rapid one-step detection of the most common pathogens in cerebrospinal fluid (CSF). However, the comprehensive performance of multiplex assays is still under evaluation. In our multisite university hospital of eastern Paris, France, ME syndromic testing has been gradually implemented since 2017 for patients with neurological symptoms presenting to an adult or pediatric emergency unit. We analyzed the results from the BioFire FilmArray ME panel versus standard routine bacteriology and virology techniques, together with CSF cytology and clinical data, over a 2.5-year period to compare the diagnostic accuracy of the FilmArray ME panel to that of the reference methods. In total, 1,744 CSF samples from 1,334 pediatric and 336 adult patients were analyzed. False-positive (mostly bacterial) and false-negative (mostly viral) cases were deciphered with the help of clinical data. The performance of the FilmArray ME panel in our study was better for bacterial detection (specificity >99%, sensitivity 100%) than viral detection (specificity >99%, sensitivity 75% for herpes simplex virus 1 [HSV-1] and 89% for enterovirus), our study being one of the largest, to date, concerning enteroviruses. The use of a threshold of 10 leukocytes/mm3 considerably increased the positive agreement between the results of the FilmArray ME panel and the clinical features, especially for bacterial pathogens, for which agreement increased from 58% to 87%, avoiding two-thirds of inappropriate testing. Based on this analysis, we propose an algorithm for the use of both syndromic and specific assays for the optimal management of suspected meningitis/encephalitis in adult and pediatric patients. IMPORTANCE Based on our comparative analysis of performances of the diagnostic assays, we propose an algorithm for the use of both syndromic and specific assays, for an optimal care of the meningitis/encephalitis threat in adult and pediatric patients.

SARS-CoV-2 B.1.1.529 (Omicron) Variant Causes an Unprecedented Surge in Children Hospitalizations and Distinct Clinical Presentation Compared to the SARS-CoV-2 B.1.617.2 (Delta) Variant.

Background: In the midst of successive waves of SARS-CoV-2 variants, the B.1.1.529 (omicron) variant has recently caused a surge in pediatric infections and hospitalizations. This study aimed to describe and compare the symptoms, explorations, treatment and evolution of COVID-19 in hospitalized children during the successive B.1.617.2 (delta) and B.1.1.529 (omicron) waves. Methods: This observational study was performed in the Pediatric Pulmonology Department of a University Hospital in Paris, France. All hospitalized children aged between 0 and 18 years who tested positive for SARS-CoV-2 using reverse transcription polymerase chain reaction (RT-PCR) in nasopharyngeal swabs from July 15th to December 15th 2021 (delta wave), and from December 15th 2021 to February 28th 2022 (omicron wave) were included. Results: In total, 53 children were included, 14 (26.4%) during the delta wave and 39 (73.6%) during the omicron wave (almost three times as many hospitalizations in half the time during the latter wave). During the omicron wave, hospitalized patients were mostly aged < 5 years (90 vs. 71% of all the children during omicron and delta waves, respectively), and tended to have fewer underlying conditions (56 vs. 79% during omicron and delta waves, respectively, p = 0.20). The omicron variant was also responsible for a different clinical presentation when compared to the delta variant, with significantly higher and often poorly tolerated temperatures (p = 0.03) and increased digestive symptoms (p = 0.01). None of the three patients who were older than 12 years were fully vaccinated. Conclusion: The dramatic increase in the hospitalization of children with COVID-19 and the modification of the clinical presentation between the latest delta and omicron waves require pediatricians to remain vigilant. It should also encourage caregivers to ensure vaccination in children older than 5 years, for whom the BNT162b2 COVID-19 vaccine has been deemed safe, immunogenic, and effective.

Retrospective observational study of the influence of the COVID-19 outbreak on infants' hospitalisation for acute bronchiolitis.

OBJECTIVES: Acute bronchiolitis is a major public health issue with high number of infants hospitalised worldwide each year. In France, hospitalisations mostly occur between October and March and peak in December. A reduction of emergency visits for bronchiolitis has been observed at onset of the COVID-19 outbreak. We aimed to assess the pandemic effects on the hospitalisations for bronchiolitis during the 2020-2021 winter (COVID-19 period) compared with three previous winters (pre-COVID-19). DESIGN: Retrospective, observational and cross-sectional study. SETTING: Tertiary university paediatric hospital in Paris (France). PARTICIPANTS: All infants aged under 12 months who were hospitalised for acute bronchiolitis during the autumn/winter seasons (1 October to 31 March) from 2017 to 2021 were included. Clinical and laboratory data were collected using standardised forms. RESULTS: During the COVID-19 period was observed, a 54.3% reduction in hospitalisations for bronchiolitis associated with a delayed peak (February instead of November-December). Clinical characteristics and hospitalisation courses were substantially similar. The differences during the COVID-19 period were: smaller proportion of infants with comorbidities (8% vs 14% p=0.02), lower need for oxygen (45% vs 55%, p=0.01), higher proportions of metapneumovirus, parainfluenzae 3, bocavirus, coronavirus NL63 and OC43 (all p≤0.01) and no influenza. The three infants positive for SARS-CoV-2 were also positive for respiratory syncytial virus, suggesting that SARS-CoV-2 alone does not cause bronchiolitis, despite previous assumptions. CONCLUSION: The dramatic reduction in infants' hospitalisations for acute bronchiolitis is an opportunity to change our future habits such as advising the population to wear masks and apply additional hygiene measures in case of respiratory tract infections. This may change the worldwide bronchiolitis burden and improve children respiratory outcomes.

Environmental severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) contamination in hospital rooms during the first and third coronavirus disease 2019 (COVID-19) waves.

We investigated the frequency, distribution, and risk factors of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) environmental contamination around infected patients during the first and third wave of the coronavirus disease 2019 pandemic. The shedding of SARS-CoV-2 in rooms of infected patients was limited in our hospital setting.

Procalcitonin at 12-36 hours of fever for prediction of invasive bacterial infections in hospitalized febrile neonates.

Aim: We aimed to investigate the performance of procalcitonin (PCT) assay between 12 and 36 h after onset of fever (PCT H12-H36) to predict invasive bacterial infection (IBI) (ie, meningitis and/or bacteremia) in febrile neonates. Methods: We retrospectively included all febrile neonates hospitalized in the general pediatric department in a teaching hospital from January 2013 to December 2019. PCT assay ≤ 0.6 ng/ml was defined as negative. The primary outcome was to study the performance of PCT H12-H36 to predict IBI. Results: Out of 385 included neonates, IBI was ascertainable for 357 neonates (92.7%). We found 16 IBI: 3 meningitis and 13 bacteremia. Sensitivity and specificity of PCT H12-H36 in the identification of IBI were, respectively, 100% [95% CI 82.9-100%] and 71.8% [95% CI 66.8-76.6%], with positive and negative predictive values of 14.3% [95% CI 8.4-22.2%] and 100% [95% CI 98.8-100%] respectively. Of the 259 neonates who had a PCT assay within the first 12 h of fever (< H12) and a PCT assay after H12-H36, 8 had IBI. Two of these 8 neonates had a negative < H12 PCT but a positive H12-H36 PCT. Conclusions: PCT H12-H36 did not miss any IBI whereas < H12 PCT could missed IBI diagnoses. PCT H12-H36 might be included in clinical decision rule to help physicians to stop early antibiotics in febrile neonates.

Diversity of the nucleic acid forms of circulating HBV in chronically infected patients and its impact on viral cycle.

BACKGROUND: Besides the prototypical hepatitis B virus (HBV) infectious particle, which contains a full-length double-stranded DNA (flDNA), additional circulating virus-like particles, which carry pregenomic RNA (pgRNA), spliced1RNA (sp1RNA) or spliced-derived DNA (defDNA) forms have been described. We aimed to determine the level of these four circulating forms in patients and to evaluate their impact on viral lifecycle. METHODS: Chronic HBV untreated patients (n = 162), included in the HEPATHER cohort, were investigated. Pangenomic qPCRs were set up to quantify the four circulating forms of HBV nucleic acids (HBVnaf). In vitro infection assays were performed to address the impact of HBVnaf. RESULTS: Hierarchical clustering individualized two clusters of HBVnaf diversity among patients: (1) cluster 1 (C1) showing a predominance of flDNA; (2) cluster 2 (C2) showing various proportions of the different forms. HBeAg-positive chronic hepatitis phase and higher viral load (7.0 ± 6.4 vs 6.6 ± 6.2 Log10 copies/ml; p < 0.001) characterized C2 compared to C1 patients. Among the different HBVnaf, pgRNA was more prevalent in C1 patients with high vs low HBV viral load (22.1% ± 2.5% vs 4.1% ± 1.8% of HBVnaf, p < 0.0001) but remained highly prevalent in C2 patients, whatever the level of replication. C2 patients samples used in infection assays showed that: (1) HBVnaf secretion was independent of the viral strain; (2) the viral cycle efficiency differed according to the proportion of HBVnaf in the inoculum, independently of cccDNA formation. Inoculum enrichment before infection suggests that pgRNA-containing particles drive this impact on viral replication. CONCLUSION: Besides the critical role of HBV replication in circulating HBVnaf diversity, our data highlight an impact of this diversity on the dynamics of viral cycle. CLINICAL TRIAL REGISTRATION: Patients were included from a prospective multicenter French national cohort (ANRS CO22 HEPATHER, NCT01953458).

Rearranged genomic RNA segments offer a new approach to the reverse genetics of rotaviruses.

Group A rotaviruses (RV), members of the Reoviridae family, are a major cause of infantile acute gastroenteritis. The RV genome consists of 11 double-stranded RNA segments. In some cases, an RNA segment is replaced by a rearranged RNA segment, which is derived from its standard counterpart by partial sequence duplication. We report here a reverse genetics system for RV based on the preferential packaging of rearranged RNA segments. Using this system, wild-type or in vitro-engineered forms of rearranged segment 7 from a human rotavirus (encoding the NSP3 protein), derived from cloned cDNAs and transcribed in the cytoplasm of COS-7 cells with the help of T7 RNA polymerase, replaced the wild-type segment 7 of a bovine helper virus (strain RF). Recombinant RF viruses (i.e., engineered monoreassortant RF viruses) containing an exogenous rearranged RNA were recovered by propagating the viral progeny in MA-104 cells, with no need for additional selective pressure. Our findings offer the possibility to extend RV reverse genetics to segments encoding nonstructural or structural proteins for which no potent selective tools, such as neutralizing antibodies, are available. In addition, the system described here is the first to enable the introduction of a mutated gene expressing a modified nonstructural protein into an infectious RV. This reverse genetics system offers new perspectives for investigating RV protein functions and developing recombinant live RV vaccines containing specific changes targeted for attenuation.

Monitoring epidemic viral respiratory infections using one-step real-time triplex RT-PCR targeting influenza A and B viruses and respiratory syncytial virus.

Rapid and specific diagnosis of influenza A/B and respiratory syncytial virus (RSV) viruses is needed for optimal management of patients with acute respiratory infections. In this study, a one-step triplex real-time RT-PCR assay was developed for rapid diagnosis of influenza A/B and RSV infections to optimize diagnosis efficiency of acute respiratory infections. Cell-culture supernatants and clinical samples were used to evaluate specificity and sensitivity of the assay. The assay was used routinely during two winter epidemics for testing respiratory specimens from 2,417 patients. The limit of detection in cell-culture supernatant was 1-10 plaque forming units/input (influenza A/B) and 2 × 10(-2) 50% tissue culture infectious dose/input (RSV). In clinical samples, the assay was as sensitive as commercial molecular assays for the detection of each influenza A/B and RSV (Flu-A/B and RSV-A/B r-gene™) individually, and far more sensitive than antigen detection. During the winter 2008-2009, the assay identified 145 RSV, 42 influenza A, and one mixed RSV-influenza A infections among 298 patients. The next winter, the assay was used in two independent hospital laboratory settings. 776 patients were tested in one hospital and 1,343 in the other, resulting in 184 and 501 RSV, 133 and 150 influenza A, and 1 and 11 mixed RSV-influenza A infections, respectively, being detected. This new user-friendly assay allows rapid (within hours), effective molecular diagnosis of single or mixed infections involving influenza A (including seasonal A H1N1 and H3N2, and A(H1N1) 2009), influenza B, and RSV(A/B). The assay is very valuable for managing patients during winter epidemics when influenza and respiratory syncytial viruses co-circulate.

Description of a late miscarriage case at 16 Weeks of Gestation associated with a SARS-CoV-2 infection.

BACKGROUND: Data about obstetric complications of maternal infection by SARS-CoV-2 remain sparse. CASE: A 40-year-old pregnant woman, gravida 3 para 1 with no previous obstetric complications, presented a late miscarriage at 16 weeks of gestation on day 9 of COVID-19 disease. The results of her nasopharyngeal swab for SARS-CoV-2, tested the same day, were negative, but the placenta was infected by SARS-CoV-2 and serology was positive 11 days later. No other obstetric or infectious cause was found to explain this outcome. CONCLUSION: This case strongly suggests that SARS-CoV-2 may lead to a late miscarriage.