Enagnon Kazali Alidjinou

Enteroviral pathogenesis of type 1 diabetes: queries and answers.

PURPOSE OF REVIEWnType 1 diabetes (T1D) results from interplay between genetic predisposition, immune system, and environmental factors. Epidemiological and experimental data strongly suggest a role for enteroviruses in the development of T1D, but a lot of controversies and unanswered questions remained. This review focuses on issues that are fueling debate.nnnRECENT FINDINGSnBeyond HLA genes, which provide genetic susceptibility for T1D, other loci have been identified to be associated with the disease. There is a link between T1D and single-nucleotide polymorphisms (SNPs) in the interferon-induced helicase 1 (IFIH1) gene that encodes melanoma differentiation-associated protein 5 (MDA5). This protein is a cytoplasmic sensor for viruses especially coxsackieviruses B, the most incriminated enteroviruses in T1D pathogenesis. Upon viral infection, MDA5 stimulates the production of mediators of the innate antiviral immune response, which is believed to play a role in a bystander activation scenario. Rare variants of IFIH1 through a lost or reduced expression of the protein are protective against T1D, whereas common IFIH1 SNPs are associated with the disease. However, a clear association has not been yet established between T1D-associated IFIH1 polymorphisms and enterovirus detection.nnnSUMMARYnLiterature have accumulated a lot of evidence supporting that enteroviruses can contribute, at least in some patients, to the pathogenesis of T1D through various mechanisms. But it is still a challenge to date to prove a causal relationship between enteroviruses and T1D. Future studies may lead to a better understanding of this relationship and ultimately can help toward disease prevention.Purpose of review Type 1 diabetes (T1D) results from interplay between genetic predisposition, immune system, and environmental factors. Epidemiological and experimental data strongly suggest a role for enteroviruses in the development of T1D, but a lot of controversies and unanswered questions remained. This review focuses on issues that are fueling debate. Recent findings Beyond HLA genes, which provide genetic susceptibility for T1D, other loci have been identified to be associated with the disease. There is a link between T1D and single-nucleotide polymorphisms (SNPs) in the interferon-induced helicase 1 (IFIH1) gene that encodes melanoma differentiation-associated protein 5 (MDA5). This protein is a cytoplasmic sensor for viruses especially coxsackieviruses B, the most incriminated enteroviruses in T1D pathogenesis. Upon viral infection, MDA5 stimulates the production of mediators of the innate antiviral immune response, which is believed to play a role in a ‘bystander activation’ scenario. Rare variants of IFIH1 through a lost or reduced expression of the protein are protective against T1D, whereas common IFIH1 SNPs are associated with the disease. However, a clear association has not been yet established between T1D-associated IFIH1 polymorphisms and enterovirus detection. Summary Literature have accumulated a lot of evidence supporting that enteroviruses can contribute, at least in some patients, to the pathogenesis of T1D through various mechanisms. But it is still a challenge to date to prove a causal relationship between enteroviruses and T1D. Future studies may lead to a better understanding of this relationship and ultimately can help toward disease prevention.

Hand, foot and mouth disease (HFMD): emerging epidemiology and the need for a vaccine strategy

Hand, foot, and mouth disease (HFMD) is a contagious viral disease and mainly affects infants and young children. The main manifestations are fever, vesicular rashes on hand, feet and buttocks and ulcers in the oral mucosa. Usually, HFMD is self-limiting, but a small proportion of children may experience severe complications such as meningitis, encephalitis, acute flaccid paralysis and neurorespiratory syndrome. Historically, outbreaks of HFMD were mainly caused by two enteroviruses: the coxsackievirus A16 (CV-A16) and the enterovirus 71 (EV-A71). In the recent years, coxsackievirus A6 and coxsackievirus A10 have been widely associated with both sporadic cases and outbreaks of HFMD worldwide, particularly in India, South East Asia and Europe with an increased frequency of neurological complications as well as mortality. Currently, there is no pharmacological intervention or vaccine available for HFMD. A formalin-inactivated EV-A71 vaccine has completed clinical trial in several Asian countries. However, this vaccine cannot protect against other major emerging etiologies of HFMD such as CV-A16, CV-A6 and CV-A10. Therefore, the development of a globally representative multivalent HFMD vaccine could be the best strategy.

Serum-Dependent Enhancement of Coxsackievirus B4-Induced Production of IFNα, IL-6 and TNFα by Peripheral Blood Mononuclear Cells

Only a few reports have been published on the interactions between Coxsackievirus B4 (CVB4) and human peripheral blood mononuclear cells (PBMC) but have not been extensively documented. Human serum containing non-neutralizing anti-CVB4 antibodies increased CVB4-induced synthesis of IFNα by PBMC. In this study, we determined if CVB4 and human serum have the ability to activate inflammatory cytokines in addition to IFNα in PBMC cultures. PBMC from healthy donors were inoculated with infectious, inactivated CVB4 or with CVB4 incubated with dilutions of human serum or polyvalent IgG with anti-CVB4 activity. Levels of IFNα, TNFα, IL-6, IL-12, IFNγ and IL-10 in the cell-free supernatants of PBMC cultures were measured using ELISA. Infection was assessed by real-time PCR. PBMC inoculated with CVB4 produced inflammatory cytokines but not IFNα. When CVB4 was incubated with serum or IgG, IFNα was detected in the culture supernatants, and high concentrations of TNFα and IL-6 were measured. The concentrations of TNFα and IL-6 were not reduced in cultures inoculated with inactivated CVB4, whereas the IgG-dependent enhancement of IFNα, IL-6 and TNFα production with inactivated virus was suppressed. The potentiation of IFNα production was associated with a high intracellular viral load. Infectious and non-infectious CVB4 can induce the production of inflammatory cytokines but not IFNα by PBMC. High levels of IFNα, in addition to TNFα and IL-6, in culture supernatants were obtained when infectious CVB4 was combined with immune serum or IgG, and they were associated with high amounts of intracellular viral RNA.

Quantification of viral DNA during HIV-1 infection: A review of relevant clinical uses and laboratory methods.

Effective antiretroviral therapy usually leads to undetectable HIV-1 RNA in the plasma. However, the virus persists in some cells of infected patients as various DNA forms, both integrated and unintegrated. This reservoir represents the greatest challenge to the complete cure of HIV-1 infection and its characteristics highly impact the course of the disease. The quantification of HIV-1 DNA in blood samples constitutes currently the most practical approach to measure this residual infection. Real-time quantitative PCR (qPCR) is the most common method used for HIV-DNA quantification and many strategies have been developed to measure the different forms of HIV-1 DNA. In the literature, several in-house PCR methods have been used and there is a need for standardization to have comparable results. In addition, qPCR is limited for the precise quantification of low levels by background noise. Among new assays in development, digital PCR was shown to allow an accurate quantification of HIV-1 DNA. Total HIV-1 DNA is most commonly measured in clinical routine. The absolute quantification of proviruses and unintegrated forms is more often used for research purposes.

Monocytes of Patients with Type 1 Diabetes Harbour Enterovirus RNA

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.

Persistent infection of human pancreatic cells with Coxsackievirus B4 is cured by fluoxetine.

Group B Coxsackieviruses (CVB) are involved in various acute clinical features and they can play a role in the development of chronic diseases like type 1 diabetes. The persistence of CVB has been described in vitro and in vivo in various models. Fluoxetine was reported to inhibit the replication of CVB1-3, which prompted us to study the in vitro antiviral activity of fluoxetine against CVB4 in models of acute infection. In addition we took advantage of a chronically CVB4-infected Panc-1 cell line to evaluate the antiviral effect of fluoxetine in a model of persistent CVB4 infection. An inhibition of the CVB4 replication was obtained when fluoxetine was added at 5.48μM to Hep-2 cell cultures. No inhibitory effect was observed when CVB4 was mixed with fluoxetine for 2h and filtered to eliminate fluoxetine before inoculation to cells, or when cells were treated up to 96h and washed before viral inoculation. Fluoxetine (5.48μM) reduced viral replication by more than 50% in acutely infected Panc-1 cell cultures. A dramatic decrease of infectious particles levels in supernatants of Panc-1 cells chronically infected with CVB4 was obtained a few days after treatment with fluoxetine and no infectious viral particle was found as soon as day 21 of treatment, and intracellular enteroviral RNA was undetectable by RT-PCR after three weeks of treatment. These data display that fluoxetine can inhibit the replication of CVB4 and can cure Panc-1 cells chronically infected with CVB4.

Human milk can neutralize Coxsackievirus B4 in vitro.

The role of enteroviruses in type 1 diabetes has long been suspected. A lower risk of type 1 diabetes is associated with breastfeeding, which could be due to a protective effect against enteroviruses. The neutralizing activity of breast milk against CVB4, a representative of enteroviruses was investigated in this study in vitro. Breast milk was cytotoxic to Hep‐2 cells up to a dilution of 1/32, whereas the aqueous fraction obtained after centrifugation was not cytotoxic; although it inhibited the cytopathic effect of CVB4 on Hep‐2 cell monolayers. The anti‐CVB4 neutralizing activity of aqueous fractions of breast milk from 49 donors living in Northern France and 15 donors living in Congo, where enteroviral infections are more prevalent, were determined. The levels of colostrum activity expressed as titre ranged from <2 to 32 in 36% of the donors from France whereas they were >128 in every donor from Congo. Pasteurized colostrum had a lower anti‐CVB4 activity compared to fresh samples (Pu2009<u20090.0001, nu2009=u200949). The treatment of colostrum samples with jacalin‐coated beads that bind specifically to human IgA, showed that IgA plays a role in anti‐CVB4 activity. There was no correlation between the neutralizing activities of breast milk and serum (Pu2009=u20090.37, nu2009=u200925). The current study showed that the variations in anti‐CVB4 activity in breast milk can be attributed to environmental and living conditions. Whether a low protective activity of breast milk against enteroviruses expose newborns to a higher risk of type 1 diabetes deserves further investigation. J. Med. Virol. 85:880–887, 2013.

Cervical samples dried on filter paper and dried vaginal tampons can be useful to investigate the circulation of high-risk HPV in Congo

BACKGROUNDnHigh-risk HPV (HR-HPV) are associated with the development of cervical cancer, the most common cancer in women in developing countries. Reliable diagnosis of HR-HPV infection combined with simple procedures to collect and store biological samples, could improve primary screening programs and vaccine strategies in these areas.nnnOBJECTIVEnTo evaluate HR-HPV detection in conventional and dried samples.nnnSTUDY DESIGNnThe presence of HR-HPV in 31 women in Republic of Congo (Central Africa) has been investigated by using standard cervical samples and dried cervical samples collected on filter paper and vaginal tampons. The detection of HPV DNA was performed in the Laboratory of virology in Lille (France) by using Hybrid capture 2 and HPV 16/18/45 Probe Set Test.nnnRESULTSn22 standard samples were found positive for the detection of HR-HPV (71%). HPV 16/18/45 was displayed in 15 out of 22 samples positive for HR-HPV (68%). The correlations between HPV detection by using standard samples and samples dried on filter paper and dried tampons were 90.3% (kappa = 0.77) and 80.6% (kappa = 0.5) respectively. The sensitivity and the specificity of HPV detection reached 91% and 89%, respectively, with samples dried on filter paper and were 86% and 67%, respectively, for dried tampons compared with standard samples.nnnCONCLUSIONnDried cervical samples and dried vaginal tampons can represent an alternative to conventional sampling to reduce barriers to large screening programs in developing countries and to facilitate storage and transport to reference centers.

Persistent coxsackievirus B4 infection induces microRNA dysregulation in human pancreatic cells

Enterovirus infections are implicated in the development of type 1 diabetes (T1D). MicroRNAs as regulators of gene expression are involved in many physiological and pathological processes. Given that viral infections dysregulate cellular microRNAs, we investigated the impact of persistent coxsackievirus B4 infection on microRNA expression of human pancreatic cells. Next-generation sequencing was used to determine microRNA expression in PANC-1 cells persistently infected (for several weeks) with coxsackievirus B4 and uninfected control cells. Target prediction restricted to T1D risk genes was performed with miRWalk2.0. Functional annotation analysis was performed with DAVID6.7. Expression of selected microRNAs and T1D risk genes was measured by quantitative reverse-transcription polymerase chain reaction. Eighty-one microRNAs were dysregulated in persistently infected PANC-1 cells. Forty-nine of the known fifty-five T1D risk genes were predicted as putative targets of at least one of the dysregulated microRNAs. Most functional annotation terms that were enriched in these 49 putative target genes were related to the immune response or autoimmunity. mRNA levels of AFF3, BACH2, and IL7R differed significantly between persistently infected cells and uninfected cells. This is the first characterization of the microRNA expression profile changes induced by persistent coxsackievirus B4 infection in pancreatic cells. The predicted targeting of genes involved in the immune response and autoimmunity by the dysregulated microRNAs as well as the dysregulated expression of diabetes risk genes shows that persistent coxsackievirus B4 infection profoundly impacts the host cell. These data support the hypothesis of a possible link between persistent coxsackievirus B4 infection and the development of T1D.

Antibodies enhance the infection of phorbol-ester-differentiated human monocyte-like cells with coxsackievirus B4

Coxsackievirus B4 (CV-B4), in presence of antibodies and through a specific viral receptor CAR and Fcγ receptors II and III, can infect monocytes which results in interferon-α synthesis. The antibody-dependent enhancement of CV-B4 infection in the human monocytic-like THP-1 cell line has been investigated. The preincubation of CV-B4 with human plasma or human polyvalent immunoglobulins enhanced the infection of phorbol-myristate-acetate (PMA)-activated THP-1 cell cultures. CV-B4 replicated in these cells as demonstrated by the intracellular detection of infectious particles, viral protein VP1 (immunofluorescence), positive and negative viral RNA (RT-PCR). The viability of infected and control cell cultures was not different up to 20 days post-infection. Activated cell cultures inoculated with CV-B4 harbored intracellular RNA up to 14 days post-infection and produced IFNα that was detected by intracellular immunofluorescence staining as soon as 4 h post-infection with a maximum at 48 h post-infection and by RT-PCR all along the experiment. Together, these data demonstrate that PMA-activated THP-1 cells can be infected with CV-B4, can produce IFNα as a result of interactions between the virus, antibodies and specific receptors. This cellular model can be used to investigate further the mechanism and the result of the antibody-dependent enhancement of CV-B4 infection.