Impact of the H274Y Substitution on N1, N4, N5, and N8 Neuraminidase Enzymatic Properties and Expression in Reverse Genetic Influenza A Viruses.

The H274Y substitution (N2 numbering) in neuraminidase (NA) N1 confers oseltamivir resistance to A(H1N1) influenza viruses. This resistance has been associated with reduced N1 expression using transfected cells, but the effect of this substitution on the enzymatic properties and on the expression of other group-1-NA subtypes is unknown. The aim of the present study was to evaluate the antiviral resistance, enzymatic properties, and expression of wild-type (WT) and H274Y-substituted NA for each group-1-NA. To this end, viruses with WT or H274Y-substituted NA (N1pdm09 or avian N4, N5 or N8) were generated by reverse genetics, and for each reverse-genetic virus, antiviral susceptibility, NA affinity (Km), and maximum velocity (Vm) were measured. The enzymatic properties were coupled with NA quantification on concentrated reverse genetic viruses using mass spectrometry. The H274Y-NA substitution resulted in highly reduced inhibition by oseltamivir and normal inhibition by zanamivir and laninamivir. This resistance was associated with a reduced affinity for MUNANA substrate and a conserved Vm in all viruses. NA quantification was not significantly different between viruses carrying WT or H274Y-N1, N4 or N8, but was lower for viruses carrying H274Y-N5 compared to those carrying a WT-N5. In conclusion, the H274Y-NA substitution of different group-1-NAs systematically reduced their affinity for MUNANA substrate without a significant impact on NA Vm. The impact of the H274Y-NA substitution on viral NA expression was different according to the studied NA.

Comparison of mortality and outcomes of four respiratory viruses in the intensive care unit: a multicenter retrospective study.

This retrospective study aimed to compare the mortality and burden of respiratory syncytial virus (RSV group), SARS-CoV-2 (COVID-19 group), non-H1N1 (Seasonal influenza group) and H1N1 influenza (H1N1 group) in adult patients admitted to intensive care unit (ICU) with respiratory failure. A total of 807 patients were included. Mortality was compared between the four following groups: RSV, COVID-19, seasonal influenza, and H1N1 groups. Patients in the RSV group had significantly more comorbidities than the other patients. At admission, patients in the COVID-19 group were significantly less severe than the others according to the simplified acute physiology score-2 (SAPS-II) and sepsis-related organ failure assessment (SOFA) scores. Using competing risk regression, COVID-19 (sHR = 1.61; 95% CI 1.10; 2.36) and H1N1 (sHR = 1.87; 95% CI 1.20; 2.93) were associated with a statistically significant higher mortality while seasonal influenza was not (sHR = 0.93; 95% CI 0.65; 1.31), when compared to RSV. Despite occurring in more severe patients, RSV and seasonal influenza group appear to be associated with a more favorable outcome than COVID-19 and H1N1 groups.

SARS-CoV-2 N-Antigen Quantification in Respiratory Tract, Plasma and Urine: Kinetics and Association with RT-qPCR Results.

Qualitative SARS-CoV-2 antigen assays based on immunochromatography are useful for mass diagnosis of COVID-19, even though their sensitivity is poor in comparison with RT-PCR assays. In addition, quantitative assays could improve antigenic test performance and allow testing with different specimens. Using quantitative assays, we tested 26 patients for viral RNA and N-antigen in respiratory samples, plasma and urine. This allowed us to compare the kinetics between the three compartments and to compare RNA and antigen concentrations in each. Our results showed the presence of N-antigen in respiratory (15/15, 100%), plasma (26/59, 44%) and urine (14/54, 28.9%) samples, whereas RNA was only detected in respiratory (15/15, 100%) and plasma (12/60, 20%) samples. We detected N-antigen in urine and plasma samples until the day 9 and day 13 post-inclusion, respectively. The antigen concentration was found to correlate with RNA levels in respiratory (p < 0.001) and plasma samples (p < 0.001). Finally, urinary antigen levels correlated with plasma levels (p < 0.001). Urine N-antigen detection could be part of the strategy for the late diagnosis and prognostic evaluation of COVID-19, given the ease and painlessness of sampling and the duration of antigen excretion in this biological compartment.

Rapid and reliable inactivation protocols for the diagnostics of emerging viruses: The example of SARS-CoV-2 and monkeypox virus.

The emergence and sustained transmission of novel pathogens are exerting an increasing demand on the diagnostics sector worldwide, as seen with the ongoing severe acute respiratory coronavirus 2 (SARS-CoV-2) pandemic and the more recent public health concern of monkeypox virus (MPXV) since May 2022. Appropriate and reliable viral inactivation measures are needed to ensure the safety of personnel handling these infectious samples. In the present study, seven commercialized diagnosis buffers, heat (56°C and 60°C), and sodium dodecyl sulfate detergent (2.0%, 1.0%, and 0.5% final concentrations) were tested against infectious SARS-CoV-2 and MPXV culture isolates on Vero cell culture. Cytopathic effects were observed up to 7 days postinoculation and viral load evolution was measured by semiquantitative polymerase chain reaction. The World Health Organization recommends an infectious titer reduction of at least 4 log10 . As such, the data show efficacious SARS-CoV-2 inactivation by all investigated methods, with >6.0 log10 reduction. MPXV inactivation was also validated with all investigated methods with 6.9 log10 reductions, although some commercial buffers required a longer incubation period to yield complete inactivation. These results are valuable for facilities, notably those without biosafety level-3 capabilities, that need to implement rapid and reliable protocols common against both SARS-CoV-2 and MPXV.

A Simple-to-Perform ifn-γ mRNA Gene Expression Assay on Whole Blood Accurately Appraises Varicella Zoster Virus-Specific Cell-Mediated Immunity After Allogeneic Hematopoietic Stem Cell Transplantation.

Herpes zoster, which is due to the reactivation of Varicella zoster virus (VZV), is a leading cause of morbidity after allogeneic hematopoietic stem cell transplantation (allo-HSCT). While cell-mediated immunity (CMI) is critical to inhibiting VZV reactivation, CMI is not routinely assessed due to a lack of reliable tests. In this study, we aimed to evaluate VZV-specific CMI among allo-HSCT recipients (n = 60) and healthy individuals (HI, n = 17) through a panel of three immune functional assays after ex vivo stimulation by VZV antigen: quantification of (i) IFN-γ release in the supernatants, (ii) T-cell proliferation after a 7-day stimulation of peripheral blood mononuclear cells (PBMC), and (iii) measurement of the ifn-γ mRNA gene expression level after 24 h of stimulation of a whole-blood sample. VZV responsiveness was defined according to IFN-γ release from VZV-stimulated PBMC. Upon VZV stimulation, we found that allo-HSCT recipients at a median time of 6 [5-8] months post-transplant had lower IFN-γ release (median [IQR], 0.34 [0.12-8.56] vs. 409.5 [143.9-910.2] pg/ml, P <.0001) and fewer proliferating T cells (0.05 [0.01-0.57] % vs. 8.74 [3.12-15.05] %, P <.0001) than HI. A subset of allo-HSCT recipients (VZV-responders, n = 15/57, 26%) distinguished themselves from VZV-non-responders (n = 42/57, 74%; missing data, n = 3) by higher IFN-γ release (80.45 [54.3-312.8] vs. 0.22 [0.12-0.42] pg/ml, P <.0001) and T-cell proliferation (2.22 [1.18-7.56] % vs. 0.002 [0.001-0.11] %, P <.0001), suggesting recovery of VZV-specific CMI. Interestingly, VZV responders had a significant fold increase in ifn-γ gene expression, whereas ifn-γ mRNA was not detected in whole blood of VZV-non-responders (P <.0001). This study is the first to suggest that measurement of ifn-γ gene expression in 24-h-stimulated whole blood could be an accurate test of VZV-specific CMI. The routine use of this immune functional assay to guide antiviral prophylaxis at an individual level remains to be evaluated.

First clinical description of letermovir resistance mutation in cytomegalovirus UL51 gene and potential impact on the terminase complex structure.

BACKGROUND: Letermovir (LMV) is a human cytomegalovirus (HCMV) terminase inhibitor indicated as prophylaxis for HCMV-positive stem-cell recipients. Its mechanism of action involves at least the viral terminase proteins pUL56, pUL89 and pUL51. Despite its efficiency, resistance mutations were characterized in vitro and in vivo, largely focused on pUL56. To date, mutations in pUL51 in clinical resistance remain to be demonstrated. METHODS: The pUL51 natural polymorphism was described by sequencing 54 LMV-naive strains and was compared to UL51 HCMV genes from 16 patients non-responding to LMV therapy (prophylaxis or curative). Recombinant viruses were built by «en-passant¼ mutagenesis to measure the impact of the new mutations on antiviral activity and viral growth. Structure prediction was performed by homology modeling. The pUL51 final-model was analyzed and aligned with the atomic coordinates of the monomeric HSV-1 terminase complex (PDB:6M5R). RESULTS: Among the 16 strains from treated-patients with LMV, 4 never described substitutions in pUL51 (D12E, 17del, A95V, V113L) were highlighted. These substitutions had no impact on viral fitness. Only UL51-A95V conferred 13.8-fold increased LMV resistance level by itself (IC50 = 29.246 ± 0.788). CONCLUSION: As an isolated mutation in pUL51 in a clinical isolate can lead to LMV resistance, genotyping for resistance should involve sequencing of the pUL51, pUL56 and pUL89 genes. With terminase modelling, we make the hypothesis that LMV could bind to domains were UL56-L257I and UL51-A95V mutations were localized.

SARS-CoV-2 Omicron Variant, Lineage BA.1, Is Associated with Lower Viral Load in Nasopharyngeal Samples Compared to Delta Variant.

OBJECTIVES: High viral load in upper respiratory tract specimens observed for Delta cases might contribute to its increased infectivity compared to the other variant. However, it is not yet documented if the Omicron variant's enhanced infectivity is also related to a higher viral load. Our aim was to determine if the Omicron variant's spread is also related to higher viral loads compared to the Delta variant. METHODS: Nasopharyngeal swabs, 129 (Omicron) and 85 (Delta), from Health Care Workers were collected during December 2021 at the University Hospital of Lyon, France. Cycle threshold (Ct) for the RdRp target of cobas® 6800 SARS-CoV-2 assay was used as a proxy to evaluate SARS-CoV-2 viral load. Variant identification was performed using a screening panel and confirmed by whole genome sequencing. RESULTS: Herein, we showed that the RT-PCR Ct values in Health Care Workers sampled within 5 days after symptom onset were significantly higher for Omicron cases than Delta cases (21.7 for Delta variant and 23.8 for Omicron variant, p = 0.008). This difference was also observed regarding patient with complete vaccination. CONCLUSIONS: This result supports the studies showing that the increased transmissibility of Omicron is related to other mechanisms than higher virus excretion.

Clinical and phylogenetic influenza dynamics for the 2019-20 season in the global influenza hospital surveillance network (GIHSN) - Pilot study.

BACKGROUND: The Global Influenza Hospital Surveillance Network (GIHSN) has operated with the aim of investigating epidemiological and clinical factors related to severe influenza-related hospitalisations. STUDY DESIGN: A common GIHSN core protocol for prospective patient enrolment was implemented. Hospital personnel completed a standardized questionnaire regarding the included patients' medical history, compiled a hospitalisation summary, collected an upper respiratory swab sample for laboratory diagnosis, and genome sequencing was performed for a subset of samples. Patient data were compared according to influenza subtype, lineage, and phylogenetic groups using the Fisher's exact test. RESULTS: From September 2019 to May 2020, 8791 patients aged ≥5 years were included. Among them, 3021 (34.4%) had a laboratory-confirmed influenza diagnosis. Influenza A(H1N1)pdm09 dominated the season among all age groups, while the B/Victoria-like lineage accounted for over half of the infections among younger age groups (5-49 years). Sequencing of the hemagglutinin segment was possible for 623 samples and revealed an influenza A and B clade frequency among severe influenza hospitalisations similar to other medically attended surveillance networks, such as the WHO GISRS. No phylogenetic clustering was observed among hemagglutinin substitutions depending on the administration of supplemental oxygen or vaccine failure. CONCLUSIONS: The GIHSN confirms its ability as an international hospital-based active surveillance network to provide valuable information on influenza infection dynamics in hospital settings. Increasing the number of participating sites and compiling more complete data, such as genome sequencing, will allow the exploration of associations between viral factors, vaccine protection, and disease severity.

A retrospective comparison of COVID-19 and seasonal influenza mortality and outcomes in the ICUs of a French university hospital.

BACKGROUND: SARS-Cov-2 (COVID-19) has become a major worldwide health concern since its appearance in China at the end of 2019. OBJECTIVE: To evaluate the intrinsic mortality and burden of COVID-19 and seasonal influenza pneumonia in ICUs in the city of Lyon, France. DESIGN: A retrospective study. SETTING: Six ICUs in a single institution in Lyon, France. PATIENTS: Consecutive patients admitted to an ICU with SARS-CoV-2 pneumonia from 27 February to 4 April 2020 (COVID-19 group) and seasonal influenza pneumonia from 1 November 2015 to 30 April 2019 (influenza group). A total of 350 patients were included in the COVID-19 group (18 refused to consent) and 325 in the influenza group (one refused to consent). Diagnosis was confirmed by RT-PCR. Follow-up was completed on 1 April 2021. MAIN OUTCOMES AND MEASURES: Differences in 90-day adjusted-mortality between the COVID-19 and influenza groups were evaluated using a multivariable Cox proportional hazards model. RESULTS: COVID-19 patients were younger, mostly men and had a higher median BMI, and comorbidities, including immunosuppressive condition or respiratory history were less frequent. In univariate analysis, no significant differences were observed between the two groups regarding in-ICU mortality, 30, 60 and 90-day mortality. After Cox modelling adjusted on age, sex, BMI, cancer, sepsis-related organ failure assessment (SOFA) score, simplified acute physiology score SAPS II score, chronic obstructive pulmonary disease and myocardial infarction, the probability of death associated with COVID-19 was significantly higher in comparison to seasonal influenza [hazard ratio 1.57, 95% CI (1.14 to 2.17); P = 0.006]. The clinical course and morbidity profile of both groups was markedly different; COVID-19 patients had less severe illness at admission (SAPS II score, 37 [28 to 48] vs. 48 [39 to 61], P < 0.001 and SOFA score, 4 [2 to 8] vs. 8 [5 to 11], P < 0.001), but the disease was more severe considering ICU length of stay, duration of mechanical ventilation, PEEP level and prone positioning requirement. CONCLUSION: After ICU admission, COVID-19 was associated with an increased risk of death compared with seasonal influenza. Patient characteristics, clinical course and morbidity profile of these diseases is markedly different.

Virological and clinical features of acute respiratory failure associated with COVID-19 in pregnancy: a case-control study.

Objective: Pregnancy is a risk factor for acute respiratory failure (ARF) following severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. We hypothesised that SARS-CoV-2 viral load in the respiratory tract might be higher in pregnant intensive care unit (ICU) patients with ARF than in non-pregnant ICU patients with ARF as a consequence of immunological adaptation during pregnancy. Design: Single-centre, retrospective observational case-control study. Setting: Adult level 3 ICU in a French university hospital. Participants: Eligible participants were adults with ARF associated with coronavirus disease 2019 (COVID-19) pneumonia. Main outcome measure: The primary endpoint of the study was viral load in pregnant and non-pregnant patients. Results: 251 patients were included in the study, including 17 pregnant patients. Median gestational age at ICU admission amounted to 28 + 3/7 weeks (interquartile range [IQR], 26 + 1/7 to 31 + 5/7 weeks). Twelve patients (71%) had an emergency caesarean delivery due to maternal respiratory failure. Pregnancy was independently associated with higher viral load (-4.6 ± 1.9 cycle threshold; P < 0.05). No clustering or over-represented mutations were noted regarding SARS-CoV-2 sequences of pregnant women. Emergency caesarean delivery was independently associated with a modest but significant improvement in arterial oxygenation, amounting to 32 ± 12 mmHg in patients needing invasive mechanical ventilation. ICU mortality was significantly lower in pregnant patients (0 v 35%; P < 0.05). Age, Simplified Acute Physiology Score (SAPS) II score, and acute respiratory distress syndrome were independent risk factors for ICU mortality, while pregnancy status and virological variables were not. Conclusions: Viral load was substantially higher in pregnant ICU patients with COVID-19 and ARF compared with non-pregnant ICU patients with COVID-19 and ARF. Pregnancy was not independently associated with ICU mortality after adjustment for age and disease severity.

Clinical and laboratory characteristics of symptomatic healthcare workers with suspected COVID-19: a prospective cohort study.

A comprehensive clinical and microbiological assessments of COVID-19 in front-line healthcare workers (HCWs) is needed. Between April 10th and May 28th, 2020, 319 HCWs with acute illness were reviewed. In addition to SARS-CoV-2 RT-PCR screening, a multiplex molecular panel was used for testing other respiratory pathogens. For SARS-CoV-2 positive HCWs, the normalized viral load, viral culture, and virus neutralization assays were performed weekly. For SARS-CoV-2 negative HCWs, SARS-CoV-2 serological testing was performed one month after inclusion. Among the 319 HCWs included, 67 (21.0%) were tested positive for SARS-CoV-2; 65/67 (97.0%) developed mild form of COVID-19. Other respiratory pathogens were found in 6/66 (9.1%) SARS-CoV-2 positive and 47/241 (19.5%) SARS-Cov-2 negative HCWs (p = 0.07). The proportion of HCWs with a viral load > 5.0 log10 cp/mL (Ct value < 25) was less than 15% at 8 days after symptom onset; 12% of HCWs were positive after 40 days (Ct > 37). More than 90% of cultivable virus had a viral load > 4.5 log10 cp/mL (Ct < 26) and were collected within 10 days after symptom onset. Among negative HCWs, 6/190 (3.2%) seroconverted. Our data suggest that the determination of viral load can be used for appreciating the infectiousness of infected HCWs. These data could be helpful for facilitating their return to work.

The Combination of Bromelain and Acetylcysteine (BromAc) Synergistically Inactivates SARS-CoV-2.

Severe acute respiratory syndrome coronavirus (SARS-CoV-2) infection is the cause of a worldwide pandemic, currently with limited therapeutic options. The spike glycoprotein and envelope protein of SARS-CoV-2, containing disulfide bridges for stabilization, represent an attractive target as they are essential for binding to the ACE2 receptor in host cells present in the nasal mucosa. Bromelain and Acetylcysteine (BromAc) has synergistic action against glycoproteins by breakage of glycosidic linkages and disulfide bonds. We sought to determine the effect of BromAc on the spike and envelope proteins and its potential to reduce infectivity in host cells. Recombinant spike and envelope SARS-CoV-2 proteins were disrupted by BromAc. Spike and envelope protein disulfide bonds were reduced by Acetylcysteine. In in vitro whole virus culture of both wild-type and spike mutants, SARS-CoV-2 demonstrated a concentration-dependent inactivation from BromAc treatment but not from single agents. Clinical testing through nasal administration in patients with early SARS-CoV-2 infection is imminent.

Characterization of SARS-CoV-2 ORF6 deletion variants detected in a nosocomial cluster during routine genomic surveillance, Lyon, France.

During routine molecular surveillance of SARS-CoV-2 performed at the National Reference Center of Respiratory Viruses (Lyon, France) (n = 229 sequences collected February-April 2020), two frameshifting deletions were detected in the open reading frame 6, at the same position (27267). While a 26-nucleotide deletion variant (D26) was only found in one nasopharyngeal sample in March 2020, the 34-nucleotide deletion (D34) was found within a single geriatric hospital unit in 5/9 patients and one health care worker in April 2020. Phylogeny analysis strongly suggested a nosocomial transmission of D34, with potential fecal transmission, as also identified in a stool sample. No difference in disease severity was observed between patients hospitalized in the geriatric unit infected with WT or D34. In vitro D26 and D34 characterization revealed comparable replication kinetics with the wild-type (WT), but differential host immune responses. While interferon-stimulated genes were similarly upregulated after infection with WT and ORF6 variants, the latter specifically induced overexpression of 9 genes coding for inflammatory cytokines in the NF-kB pathway, including CCL2/MCP1, PTX3, and TNFα, for which high plasma levels have been associated with severe COVID-19. Our findings emphasize the need to monitor the occurrence of ORF6 deletions and assess their impact on the host immune response.

Performance Assessment of SARS-CoV-2 PCR Assays Developed by WHO Referral Laboratories.

A reliable diagnostic assay is crucial to early detect new COVID-19 cases and limit severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) transmission. Since the onset of the COVID-19 pandemic, the World Health Organization has published several diagnostic molecular approaches developed by referral laboratories, including Charité (Germany), HKU (Hong Kong), China CDC (China), US CDC (United States), and Institut Pasteur, Paris (France). We aimed to compare the sensitivity and specificity of these different RT-PCR assays using SARS-CoV-2 cell culture supernatants and clinical respiratory samples. Overall, the different RT-PCR assays performed well for SARS-CoV-2 detection and were all specific except the N Charité (Germany), and N2 US CDC (United States) assays. RdRp Institut Pasteur (IP2, IP4), N China CDC, and N1 US CDC were found to be the most sensitive assays. The data presented herein are of prime importance to facilitate the equipment choice of diagnostic laboratories, as well as for the development of marketed tests.

Phenotypic testing of patient herpes simplex virus type 1 and 2 isolates for acyclovir resistance by a novel method based on real-time cell analysis.

BACKGROUND: Acyclovir (ACV) is the most commonly used drug for herpes simplex virus (HSV) infection therapy. Prolonged antiviral therapy or prophylaxis in immunocompromised patients may promote the development of drug-resistant strains. Due to the high polymorphism in genes involved in drug resistance, phenotypic methods, although work-intensive, are still required to test drug susceptibility. Real-time cell analysis (RTCA) based methods could offer a rapid and less labor-intensive alternative for phenotypic testing of ACV resistance. OBJECTIVE: To investigate the utility of a new RTCA based assay (RTCAA) to test acyclovir susceptibility of HSV clinical isolates. STUDY DESIGN: Four reference strains and 93 clinical isolates (60 HSV-1 and 33 HSV-2) were tested by RTCAA. In the presence of ACV concentrations from 2.2 to 140.8 µM, Vero cells were infected with different virus dilutions. IC50 values were calculated by dose-response curve (DRC) with area-under-curve (AUC) method. The reference strains and 22 clinical isolates were additionally tested by dye-uptake assay, and IC50 values of both methods were compared. RESULTS: IC50 values from RTCAA and dye-uptake assays were positively correlated (Spearman's rho = 0.897, p < 0.001) and quantitatively agreed (Bland-Altman plot). Based on a cut-off of 4 µM for HSV-1 and 13 µM for HSV-2, 87 isolates were classified as ACV-sensitive and 6 isolates as ACV-resistant. The reference strains showed the expected results of ACV susceptibility. CONCLUSION: RTCAA agrees well with the dye-uptake assay. Compared with other phenotypic methods, RTCAA requires less manipulation, reduces the workload and the turnaround time, and appears to be an objective and reliable method to test ACV susceptibility.