Clofoctol inhibits SARS-CoV-2 replication and reduces lung pathology in mice.

Drug repurposing has the advantage of shortening regulatory preclinical development steps. Here, we screened a library of drug compounds, already registered in one or several geographical areas, to identify those exhibiting antiviral activity against SARS-CoV-2 with relevant potency. Of the 1,942 compounds tested, 21 exhibited a substantial antiviral activity in Vero-81 cells. Among them, clofoctol, an antibacterial drug used for the treatment of bacterial respiratory tract infections, was further investigated due to its favorable safety profile and pharmacokinetic properties. Notably, the peak concentration of clofoctol that can be achieved in human lungs is more than 20 times higher than its IC50 measured against SARS-CoV-2 in human pulmonary cells. This compound inhibits SARS-CoV-2 at a post-entry step. Lastly, therapeutic treatment of human ACE2 receptor transgenic mice decreased viral load, reduced inflammatory gene expression and lowered pulmonary pathology. Altogether, these data strongly support clofoctol as a therapeutic candidate for the treatment of COVID-19 patients.

Antibody-dependent enhancement and neutralization against CVB4 investigated in vitro and in silico through an agent-based model.

The infection with coxsackievirus B4 (CVB4) can be enhanced in vitro by antibodies directed against the viral capsid protein VP4. In peripheral blood mononuclear cells, antibody-dependent enhancement (ADE) of CVB4 infection leads to the production of interferon alpha (IFN-α). To investigate ADE of CVB4-induced production of IFN-α, an agent-based model was constructed with enhancing and neutralizing antibodies. The model recapitulates viral neutralization and ADE in silico. The enhancing and neutralizing activities of serum samples were evaluated in vitro to confront the model predictions with experimental results. Increasing the incubation time of CVB4 with serum samples improves virus neutralization in silico as well as in vitro. It also results in ADE at lower antibody numbers in silico, which is confirmed in vitro with IFN-α production at lower serum concentrations. Furthermore, incubation of CVB4 with serum at a low temperature does not induce IFN-α production in vitro. Thus, taken together our results suggest that enhancing antibodies bind cryptic epitopes, more accessible with longer incubation time and at higher temperature due to changes in capsid conformation, consistent with previous results indicating that enhancing antibodies are anti-VP4 antibodies.

Combating coxsackievirus B infections.

Coxsackieviruses B (CVB) are small, non-enveloped, single-stranded RNA viruses belonging to the Enterovirus genus of the Picornaviridae family. They are common worldwide and cause a wide variety of human diseases ranging from those having relatively mild symptoms to severe acute and chronic pathologies such as cardiomyopathy and type 1 diabetes. The development of safe and effective strategies to combat these viruses remains a challenge. The present review outlines current approaches to control CVB infections and associated diseases. Various drugs targeting viral or host proteins involved in viral replication as well as vaccines have been developed and shown potential to prevent or combat CVB infections in vitro and in vivo in animal models. Repurposed drugs and alternative strategies targeting miRNAs or based on plant extracts and probiotics and their derivatives have also shown antiviral effects against CVB. In addition, clinical trials with vaccines and drugs are underway and offer hope for the prevention or treatment of CVB-induced diseases.

Oropharyngeal samples versus lesion specimens at diagnosis in patients infected with monkeypox virus in Northern France.

The ongoing outbreak of monkeypox virus (MPXV) is the largest one in historically non-endemic countries. Early reports described atypical epidemiological and clinical presentations. We investigated MPXV DNA detection in oropharyngeal samples (OPS), and compared the viral load to that in lesion samples at diagnosis in patients infected with MPXV. We retrospectively included patients suspected to have monkeypox in Northern France, who underwent a MPXV PCR in the Virology Laboratory, University Hospital of Lille, from May 23 to August 18, 2022. Overall, a total of 228 patients (376 samples) were included. A positive result in at least one sample was found in 138 patients (60.5%). We compared PCR results between OPS and lesion samples (i.e., cutaneous or anal/rectal samples) in patients with both samples. A positive result in OPS was observed in 54 out of 60 patients (90%). The viral load in OPS (median Ct value = 29.5; interquartile range [IQR] = 24.7-34) was significantly lower than that in lesion samples (median Ct  value = 17.8; IQR = 16.3 and 19.7) (p < 0.0001). This report shows that pharyngeal sampling does not bring additional information for the initial diagnosis in patients presenting with typical lesions.

The role of the surface ligand on the performance of electrochemical SARS-CoV-2 antigen biosensors.

Point-of-care (POC) technologies and testing programs hold great potential to significantly improve diagnosis and disease surveillance. POC tests have the intrinsic advantage of being able to be performed near the patient or treatment facility, owing to their portable character. With rapid results often in minutes, these diagnostic platforms have a high positive impact on disease management. POC tests are, in addition, advantageous in situations of a shortage of skilled personnel and restricted availability of laboratory-based analytics. While POC testing programs are widely considered in addressing health care challenges in low-income health systems, the ongoing pandemic of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections could largely benefit from fast, efficient, accurate, and cost-effective point-of-care testing (POCT) devices for limiting COVID-19 spreading. The unrestrained availability of SARS-CoV-2 POC tests is indeed one of the adequate means of better managing the COVID-19 outbreak. A large number of novel and innovative solutions to address this medical need have emerged over the last months. Here, we critically elaborate the role of the surface ligands in the design of biosensors to cope with the current viral outbreak situation. Their notable effect on electrical and electrochemical sensors' design will be discussed in some given examples. Graphical abstract.

[Coxsackievirus B infection and pathogenesis of type 1 diabetes]. / Infection à coxsackievirus B et pathogenèse du diabète de type 1.

Epidemiological and experimental studies suggest that enteroviruses (EV) and particularly coxsackieviruses B (CVB) are likely to trigger or accelerate the onset of islet autoimmunity and the development of type 1 diabetes (T1D) in genetically susceptible individuals. Several mutually non-exclusive mechanisms have been proposed to explain the involvement of CVB in the pathogenesis of T1D. CVB can infect and persist in the intestine, thymic cells, monocytes/macrophages, ductal cells and pancreatic ß-cells, which leads to structural or functional alterations of these cells. A chronic inflammatory response and disruption of tolerance towards ß-cells due to CVB infections are able to promote the recruitment and activation of pre-existing autoreactive T-cells and the destruction of ß-cells. Vaccine or therapeutic strategies to control EV infections have been developed and open perspectives for the prevention or treatment of T1D.

Assessment of intra-sample variability in HIV-1 DNA drug resistance genotyping.

BACKGROUND AND OBJECTIVES: HIV-1 drug resistance testing can be performed in proviral DNA. The non-homogenous distribution of viral variants in cells can impact the performance of this method. We assessed the variability of HIV-1 DNA genotyping results in the same blood sample using a next-generation sequencing (NGS) method. METHODS: For each included patient, a blood sample from a single venipuncture was split into five 1 mL aliquots, which were independently tested in the same run. HIV-1 DNA was quantified in blood samples using real-time PCR, and NGS was performed with the Sentosa platform combined with the Sentosa SQ HIV genotyping Assay. RESULTS: A total of 60 aliquots from 12 samples (12 patients) were tested. The median age was 45.50 years old, and all patients were treated with antiretrovirals. A significant variability can sometimes be observed in HIV-1 DNA quantification between aliquots from the same sample, with a coefficient of variation ranging from 23% to 89%. The analysis of resistance-associated mutations (RAMs) with a 20% cut-off found some discordances in RAMs profile between aliquots from the same sample for 5, 3 and 3 patients in the reverse transcriptase, protease and integrase genes, respectively. The analysis with a lower cut-off (10%) showed additional mutations, but did not improve the intra-sample concordance. CONCLUSIONS: There is an intra-sample variability in HIV-1 DNA resistance test results, and repetition may sometimes bring additional information, but the extent of its clinical impact still requires further investigation.

Pancreatic beta cells persistently infected with coxsackievirus B4 are targets of NK cell-mediated cytolytic activity.

It has been suggested that the persistence of coxsackieviruses-B (CV-B) in pancreatic beta cells plays a role in the pathogenesis of type 1 diabetes (T1D). Yet, immunological effectors, especially natural killer (NK) cells, are supposed to clear virus-infected cells. Therefore, an evaluation of the response of NK cells to pancreatic beta cells persistently infected with CV-B4 was conducted. A persistent CV-B4 infection was established in 1.1B4 pancreatic beta cells. Infectious particles were found in supernatants throughout the culture period. The proportion of cells containing viral protein VP1 was low (< 5%), although a large proportion of cells harbored viral RNA (around 50%), whilst cell viability was preserved. HLA class I cell surface expression was downregulated in persistently infected cultures, but HLA class I mRNA levels were unchanged in comparison with mock-infected cells. The cytolytic activities of IL-2-activated non-adherent peripheral blood mononuclear cells (PBMCs) and of NK cells were higher towards persistently infected cells than towards mock-infected cells, as assessed by an LDH release assay. Impaired cytolytic activity of IL-2-activated non-adherent PBMCs from patients with T1D towards infected beta cells was observed. In conclusion, pancreatic beta cells persistently infected with CV-B4 can be lysed by NK cells, implying that impaired cytolytic activity of these effector cells may play a role in the persistence of CV-B in the host and thus in the viral pathogenesis of T1D.

Repeated viral meningitis in a newborn.

Human enteroviruses (EV) are the most common cause of viral meningitis in children. Human parechoviruses (HPeV) are increasingly being recognized as a cause of central nervous system (CNS) infections and sepsis-like disease in children. Both viruses belong to Picornaviridae family. The clinical picture in EV and HPeV infections is usually nonspecific. Therefore, molecular detection of both viruses is needed for etiological diagnosis. In this case report, we describe and discuss clinical and laboratory findings of two consecutive episodes of viral meningitis caused by EV and HPeV, respectively, occurring in the first month of a newborn's life.

Routine drug resistance testing in proviral HIV-1 DNA: Prevalence of stop codons and hypermutation, and associated factors.

We investigated the presence of stop codons (SC) and/or hypermutation (HM) in HIV-1 DNA sequences generated for routine drug resistance testing in proviral HIV-1 DNA, and sought for associated factors. At least one SC was identified in 6.2% of HIV-1 DNA sequences, among which 54.8% were hypermutated. The defective virus group (SC w/o HM) was similar to the non-SC group regarding the characteristics of HIV-1 infection, and before drug exposure. In addition, the HIV-1 DNA levels were not different between both groups. Sequences with SC/HM displayed a higher proportion of RAMs. The impact of the SC/HM associated RAMs on clinical responses requires further investigation.

Aqueous extracts of Syzygium brazzavillense can inhibit the infection with coxsackievirus B4 in vitro.

Traditional practitioners commonly use plant crude extracts to treat various diseases in patients with symptoms that can be seen during enterovirus infections. In this study, the antienteroviral activity of medicinal plants from the Republic of Congo has been evaluated in vitro. Through an ethnopharmacological approach, seven plants grouped into six families were identified. Aqueous and organic extracts of various organs from these plants were prepared. The organic extracts at subcytotoxic concentrations did not inhibit the cytopathic effect (CPE) induced by coxsackievirus (CV)B1-5, CVA6, poliovirus type 1, and enterovirus 71. The aqueous extract of Syzygium brazzavillense, but not those of other plants, inhibited the CPE induced by CVB3 and CVB4 at 30 µg/mL (CC50 ; 2800 µg/mL, IC50 ; 0.8 µg/mL) and by CVB2 and poliovirus type 1 at higher concentrations. When aqueous extract of this plant was mixed with CVB4, the replication of the virus was inhibited. In conclusion, aqueous extracts of Syzygium brazzavillense can inhibit the infection with CVB4 and other enteroviruses in vitro. The present ethnopharmacological investigation helped to identify a plant with potential properties useful to combat enterovirus infections.

miRNAs in enterovirus infection.

During the last years, it has become evident that miRNAs are important players in almost all physiological and pathological processes, including viral infections. Enterovirus infections range from mild to severe acute infections concerning several organ systems and are also associated with chronic diseases. In this review, we summarize the findings on the impact of acute and persistent enterovirus infection on the expression of cellular miRNAs. Furthermore, the currently available data on the regulation of cellular or viral targets by the dysregulated miRNAs are reviewed. Finally, a translational perspective, namely the use of miRNAs as biomarkers of enterovirus infection and as antiviral strategy is discussed.

Serum-derived IgG from coxsackievirus A6-infected patients can enhance the infection of peripheral blood mononuclear cells with coxsackievirus A6.

Coxsackievirus A6 (CV-A6) has recently emerged as an enterovirus causing Hand Foot and Mouth Disease with severe complications. The pathogenic mechanisms of CV-A6- associated Hand foot and Mouth disease are largely unknown. In this study, it was investigated whether serum and IgG from patients with CV-A6 infection can enhance the infection of PBMC with the virus. Serum samples were obtained from five children with CV-A6 infection confirmed by RT-PCR and seven controls. IgG was isolated from serum by using affinity chromatography columns. CV-A6 was incubated with serum or IgG from controls and patients then the mixtures were added to PBMC cultures. The levels of IFNα in supernatants were measured by ELISA, and the levels of intracellular viral RNA were measured by RT-qPCR. It has been observed that there is an anti-CV-A6 enhancing activity in serum and serum-derived immunoglobulin G of children with CV-A6 infection but not in those of uninfected controls. Whether this activity has implications in the pathogenesis of CV-A6 associated diseases should be investigated.

Resistance of Enteric Viruses on Fomites.

Human enteric viruses are associated with several clinical features, especially gastroenteritis. Large amounts of these viruses can be released in the environment and spread to people. Enteric viruses are nonenveloped viruses and have displayed good survival in the environment. They can be significantly resistant in food and water but also on fomites, and this is thought to play a role in transmission, leading to sporadic cases or outbreaks. The survival of enteric viruses on fomites relies on many factors including the virus itself, fomite properties, and extrinsic environmental factors such as temperature or relative humidity. Several reports in the literature have found an association with gastroenteritis cases or outbreaks and fomites naturally contaminated by enteric viruses. However, the study of virus survival following natural contamination is challenging, and most published studies are laboratory based, using experimental contamination. In addition, recent and detailed data on the resistance of each of the main enteric viruses on fomites are scarce. Many approaches, both physical and chemical, can be used to inactivate enteric viruses, the efficacy of which depends on the virus and the disinfection conditions.

Monocytes of Patients with Type 1 Diabetes Harbour Enterovirus RNA.

BACKGROUND: Intracellular enterovirus (EV) RNA was detected in blood of patients with type 1 diabetes (T1D). The presence of EV RNA in subsets of peripheral blood mononuclear cells (PBMCs) of patients, and the in vitro infection of these cells with an EV, was investigated. MATERIALS AND METHODS: Blood was collected from 42 patients with T1D, PBMCs were isolated and monocytes were purified. Interferon alpha (IFNα) mRNA and EV RNA were investigated using RT-PCR. Levels of IFNα in plasma were measured using an immunoassay. Cells were inoculated with Coxsackievirus B4 (CBV4) in vitro, and infection was assessed by indirect immunofluorescence (IFI). RESULTS: Interferon alpha mRNA was detected in blood and in monocytes of 12 of 42 patients with T1D, but not in monocyte-depleted PBMCs of the same individuals. Significant plasma levels of IFNα (≥ 5 IU/mL) were found in six patients. EV RNA was detected in whole blood and in monocytes of seven patients and negative-strand EV RNA was found in monocytes of 6 of them. When monocytes of patients with IFNα and/or EV RNA in their blood were inoculated with CVB4, the proportion of cells stained by an anti-VP1 antibody was 8.8 ± 1%, whereas no VP1 was detected in the monocytes of IFNα, EV RNA negative patients. Nevertheless, when CBV4 was mixed with plasma, VP1 was detected in monocytes of all patients with T1D (staining ranging from 12 to 36%). CONCLUSIONS: Our data indicate that monocytes of patients with T1D can harbor EV RNA and IFNα mRNA and can be infected with an EV in vitro.

The relationship between SARS-CoV-2 infection and type 1 diabetes mellitus.

Environmental factors, in particular viral infections, are thought to have an important role in the pathogenesis of type 1 diabetes mellitus (T1DM). The COVID-19 pandemic reinforced this hypothesis as many observational studies and meta-analyses reported a notable increase in the incidence of T1DM following infection with SARS-CoV-2 as well as an association between SARS-CoV-2 infection and the risk of new-onset T1DM. Experimental evidence suggests that human ß-cells express SARS-CoV-2 receptors and that SARS-CoV-2 can infect and replicate in ß-cells, resulting in structural or functional alterations of these cells. These alterations include reduced numbers of insulin-secreting granules, impaired pro-insulin (or insulin) secretion, and ß-cell transdifferentiation or dedifferentiation. The inflammatory environment induced by local or systemic SARS-CoV-2 infection might result in a set of signals (such as pro-inflammatory cytokines) that lead to ß-cell alteration or apoptosis or to a bystander activation of T cells and disruption of peripheral tolerance that triggers autoimmunity. Other mechanisms, such as viral persistence, molecular mimicry and activation of endogenous human retroviruses, are also likely to be involved in the pathogenesis of T1DM following SARS-CoV-2 infection. This Review addresses the issue of the involvement of SARS-CoV-2 infection in the development of T1DM using evidence from epidemiological, clinical and experimental studies.

Frequency and burden of disease for SARS-CoV-2 and other viral respiratory tract infections in children under the age of 2 months.

OBJECTIVE: To evaluate the frequency and burden of disease of SARS-CoV-2 and other respiratory viruses in children under the age of 2 months. METHODS: A retrospective, cross-sectional, single-center study was conducted between March 2021 and February 2022. All children under the age of 2 months and tested for SARS-CoV-2 were included. The frequency of SARS-CoV-2, of other respiratory viruses and the burden of disease caused by SARS-CoV-2 and other respiratory viruses were evaluated. RESULTS: Seven hundred and twenty-seven children with an RT-PCR test for SARS-CoV-2 were included (mean age: 0.9 months (±0.6); boys: 57%); 514 (71%) in the emergency room and 213 (29%) in hospital. Among them, 62 (8.5%) had a positive RT-PCR test for SARS-CoV-2, more often in the Omicron period (23%) than in the Alpha period (4%). Of the 565 (78%) with a multiplex RT-PCR test for other viruses, 325 (58%) were positive. Children with a positive SARS-CoV-2 were less likely to have required respiratory support (p = 0.001), enteral nutrition (p = 0.03), or intensive care admission (p = 0.01) and had a shorter hospital stay than children with other respiratory viruses (5 days vs. 7 days, p = 0.007). CONCLUSION: In this young population of children, SARS-CoV-2 infection was less frequent and less severe than other viral respiratory infections.