Human Endogenous Retroviruses and Type 1 Diabetes.

PURPOSE OF THE REVIEW: The aim of this review is to discuss recent data pointing at an involvement of human endogenous retroviruses (HERVs) in type 1 diabetes (T1D) onset and progression. RECENT FINDINGS: The envelope protein of HERV-W family, named HERV-W-Env, was detected in pancreata from T1D patients and was shown to display pro-inflammatory properties and direct toxicity toward pancreatic beta cells. The etiopathogenesis of T1D remains elusive, even if conventional environmental viral infections have been recurrently involved. Nonetheless, a new category of pathogens may provide the missing link between genetic susceptibility and environmental factors long thought to contribute to T1D onset. A number of studies have now shown that HERV sequences, which are normally inactivated or repressed in the human genome, could be activated by environmental viruses. Thus, if similarly activated by viruses associated with T1D, disregarded HERV genes may underlie T1D genetic susceptibility. Moreover, once expressed, HERV elements may display broad pathogenic properties, which identify them as potential new therapeutic targets.

RNA and DNA Sanger sequencing versus next-generation sequencing for HIV-1 drug resistance testing in treatment-naive patients.

Background: Sanger sequencing of plasma RNA is the standard method for HIV-1 drug resistance testing in treatment-naive patients, but is limited by the non-detection of resistance-associated mutations (RAMs) with prevalence below approximately 20%. Objectives: We compared RNA and DNA Sanger sequencing (RSS and DSS) with RNA next-generation sequencing (NGS) for RAM detection in HIV-1 reverse transcriptase (RT), protease (PR) and integrase (IN) genes. Methods: Sanger sequencing was performed on RNA and DNA, following the recommendations of the French Agency for AIDS Research (ANRS). NGS was performed on RNA using the HIV-1 Drug Resistance Assay, v. 3.0 (Roche) on the 454 GS Junior sequencer. The IAS-USA list was used to identify RAMs. ANRS, Rega and Stanford algorithms were used for drug resistance interpretation. Results: The study included 48 ART-naive patients. The number of patients with at least one major RAM was 3, 3, 4 and 8 when using RSS, DSS, NGS 20% and NGS 5%, respectively. Numerous minor mutations were detected in patients, especially in the protease gene. None of the methods detected any major mutation in the integrase gene. Overall, the mutation detection rate was similar between RSS and DSS, and higher with NGS 20%. Differences in drug resistance interpretation were found between algorithms. No impact of the minority RAMs detected by NGS was found on the short-term treatment outcome. Conclusions: DSS does not clearly improve the detection of RAMs in ART-naive patients, as compared with RSS. NGS allows detection of additional minority RAMs; however, their clinical relevance requires further investigation.

In-utero coxsackievirus B4 infection of the mouse thymus.

Type B coxsackievirus (CV-B) infections are involved frequently in the triggering of several autoimmune diseases such as myocarditis, dilated cardiomyopathy, pericarditis, pancreatitis, type 1 diabetes, encephalitis, thyroiditis or Sjögren's syndrome. Serological and virological evidence suggests that maternal infections during pregnancy can play a role in the appearance of these diseases in offspring. The current study aims to explore the effect of an in-utero CV-B infection on the fetal thymus, the central site for programming immunological self-tolerance. In this perspective, female Swiss albino mice were inoculated intraperitoneally or orally with the diabetogenic CV-B4 E2 strain at gestational days 10 or 17. Offspring were killed at different post-inoculation times, and their thymuses were analysed for evidence of infection and alterations in thymic T cell subsets. In-utero CV-B infection of the thymus was demonstrated during the course of vertical transmission, as attested by viral RNA and infectious virus detection in most analysed samples. No histopathological changes were evident. Thymic T cells were not depleted, despite being positive for viral RNA. As evidenced by flow cytometry analysis, CV-B infection of the fetal thymus induced significant changes of thymic T cell populations, particularly with maternal inoculation at gestational day 10. Altogether, these findings suggest that CV-B infection of the fetal thymus may play an important role in the genesis of autoimmune diseases.

Marker of coxsackievirus-B4 infection in saliva of patients with type 1 diabetes.

BACKGROUND: Coxsackieviruses B (CV-B) are enteroviruses that have been reported to play a role in the pathogenesis of type 1 diabetes. Enteroviral RNA was detected in the gut mucosa of patients. The mucosal immunity is an interconnected network; therefore, the response to enteroviruses possibly present in the gastrointestinal mucosa can be reflected by specific antibodies in the saliva. In the present study, the anti-CV-B neutralizing activity of saliva samples from patients with type 1 diabetes was investigated. METHODS: Saliva samples were collected from patients and controls of 3 countries, and plasma was obtained from some of them. The anti-CV-B activity of clinical samples was determined by neutralization of the cytopathic effect induced by challenging viruses in vitro and expressed as titre value. RESULTS: Overall prevalence and levels of anti-CV-B4 activity of saliva were higher in patients (n = 181) than in controls (n = 135; P = .0002; titre values ≥ 16: odds ratio = 4.22 95% CI: 1.90-9.38 P = .0002). It has been shown that IgA1 played a role in this activity. There was no correlation between the saliva and the plasma anti-CV-B4 neutralizing activity. The neutralizing activity of saliva against CV-B1, CV-B2, CV-B3, and CV-B5 existed rarely, if at all. Increased levels of anti-CV-B4 activity were observed all along a 4 year follow-up period in patients but not in matched controls (P = .01). CONCLUSION: There is an anti-CV-B4 activity in saliva of patients with type 1 diabetes that may be a useful marker to study the role of CV-B in the pathogenesis of the disease.

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.

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.

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 (P < 0.0001, n = 49). 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 (P = 0.37, n = 25). 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.

Immunology in the clinic review series; focus on type 1 diabetes and viruses: role of antibodies enhancing the infection with Coxsackievirus-B in the pathogenesis of type 1 diabetes.

Type 1 diabetes results from an interaction between genetic and environmental factors. Coxsackieviruses B (CV-B) are major environmental candidates, as suggested by epidemiological and experimental studies. The mechanisms leading to the disease involve interactions between the virus, host target tissue (pancreas) and the immune system. The infection of target cells with viruses can be prevented by antibodies. Conversely, the infection can be enhanced by antibodies. The antibody-dependent enhancement (ADE) of infection has been described with various viruses, especially Picornaviruses. In mice infected with CV-B3 this phenomenon resulted in an extended inflammatory reaction and myocarditis. In the human system non-neutralizing antibodies can increase the infection of monocytes with CV-B4 and stimulate the production of interferon (IFN)-α by these cells in vitro. CV-B4/immunoglobulin (Ig)G immune complexes interacted with a specific viral receptor [Coxsackievirus and adenovirus receptor (CAR)] and with IgG Fc fraction receptors (FcγRII and FcγRIII) at the surface of monocytes. The virus-antibody complexes are internalized (CAR) and receptors for the Fc of IgG (FcγRII and FcγRIII). Such antibodies have been detected in patients with type 1 diabetes and they could be responsible for the presence of enteroviral RNA and IFN-α in peripheral blood mononuclear cells (PBMC) of these individuals. The target of enhancing antibodies has been identified as the VP4 protein, which allowed the detection of these antibodies by enzyme-linked immunosorbent assay (ELISA). It cannot be excluded that antibodies enhancing the infection with CV-B may play a role in the pathogenesis of type 1 diabetes, induced or aggravated by these viruses. They can cause a viral escape from the immune response and may participate in the spreading of viruses to ß cells. Whether enhancing antibodies raised against VP4 can play a role in iterative homologous and/or heterologous CV-B infections and in the persistence of viruses within the host deserves further study.

Immunology in the clinic review series; focus on type 1 diabetes and viruses: enterovirus, thymus and type 1 diabetes pathogenesis.

Thymus dysfunction, especially immune suppression, is frequently associated with various virus infections. Whether viruses may disturb the thymus function and play a role in the pathogenesis of autoimmune diseases is an open issue. Enteroviruses, especially Coxsackievirus B4 (CV-B4), have been largely suggested as potential inducers or aggravating factors of type 1 diabetes (T1D) pathogenesis in genetically predisposed individuals. Several pathogenic mechanisms of enterovirus-induced T1D have been suggested. One of these mechanisms is the impairment of central self-tolerance due to viral infections. Coxsackievirus-B4 is able to infect murine thymus in vitro and in vivo and to infect human thymus in vitro. Thymic epithelial cells and thymocytes are targets of infection with this virus, and several abnormalities, especially disturbance of maturation/differentiation processes, were observed. Altogether, these data suggest that CV-B infection of thymus may be involved in the pathogenesis of T1D. Further investigations are needed to explore this hypothesis.

High incidence of Epstein-Barr virus, cytomegalovirus, and human-herpes virus-6 reactivations in critically ill patients with COVID-19.

BACKGROUND: Systemic reactivation of herpesviruses may occur in intensive care unit (ICU) patients and is associated with morbidity and mortality. Data on severe Coronavirus disease-19 (COVID-19) and concomitant reactivation of herpesviruses are lacking. METHODS: We selected patients admitted to ICU for confirmed COVID-19 who underwent systematic testing for Epstein-Barr virus (EBV), cytomegalovirus (CMV) and human-herpes virus-6 (HHV-6) DNAemia while in the ICU. We retrospectively analysed frequency, timing, duration and co-occurrence of viral DNAemia. RESULTS: Thirty-four patients were included. Viremia with EBV, CMV, and HHV-6 was detected in 28 (82%), 5 (15%), and 7 (22%) patients, respectively. EBV reactivation occurred early after ICU admission and was associated with longer ICU length-of-stay. CONCLUSIONS: While in the ICU, critically ill patients with COVID-19 are prone to develop reactivations due to various types of herpesviruses.

Coxsackievirus B4 and type 1 diabetes pathogenesis: contribution of animal models.

The role of enteroviruses, in particular type B coxsackieviruses (CV-B), in type 1 diabetes (T1D) pathogenesis is supported by epidemiological, clinical and experimental observations.The investigation of T1D pathogenesis benefits from the contribution of animal models called spontaneously diabetic. Among these animals the non-obese diabetic (NOD) mouse and the bio-breeding diabetes-prone (BBDP) rat present a genetic susceptibility manifested by the expression of an autoimmune diabetes similar to the pathology observed in human beings. Other models whose genetic predisposition is less known are of considerable contribution as well. Numerous major observations relative to several aspects of T1D pathogenesis in the context of CV-B infections, such as susceptibility, diabetogenicity, pancreatotropism, mechanisms of beta cells destruction and others, have been deduced thanks to investigations with animal models. Despite their limits, these models are necessary in improving our knowledge of the role of enteroviruses, like CV-B4, in the pathogenesis of T1D, and the recent advances ensuing from their contribution may have important therapeutic and preventive spin-offs.

[When an enterovirus emerges]. / Quand un entérovirus émerge.

Most of enterovirus infections are benign and the rate of mortality is low in countries with temperate climates. But since the late 1990s, Enterovirus 71 (EV-71) has become much more aggressive in Asian countries, with the outcome of a neurogenic pulmonary oedema syndrome and it is responsible for huge epidemics. The virological diagnosis rely upon viral isolation and identification by sero-neutralization, and upon the detection of specific IgM by ELISA and viral RNA by RT-PCR. There is no specific treatment to fight this virus, but innovative strategies, especially based on interfering RNA, are under investigation.

Role of coxsackievirus B4 in the pathogenesis of type 1 diabetes.

Environmental factors, especially viruses, are thought to play an important role in the initiation or acceleration of the pathogenesis of type 1 diabetes (T1D). Data from retrospective and prospective epidemiological studies strongly suggest that enteroviruses, such as coxsackievirus B4 (CV-B4), may be associated with the development of T1D. It has also been shown that enterovirus infections are significantly more prevalent in at-risk individuals such as the siblings of diabetic patients, when they develop anti-beta-cell autoantibodies or T1D, and in recently diagnosed diabetic patients, compared with control subjects. The isolation of CV-B4 from the pancreas of diabetic patients supports the hypothesis of a relationship between the virus and the disease. Furthermore, studies performed in vitro and in vivo in animal models have increased our knowledge of the role of CV-B4 in T1D by helping to clarify the pathogenic mechanisms of the infection that can lead to beta-cell destruction, including direct virus-induced beta-cell lysis, molecular mimicry, 'bystander activation' and viral persistence. The role of enteroviruses as the sole agents in T1D, and a causal link between these agents and T1D, have not yet been established, although arguments that support such a role for these viruses in the pathogenesis of the disease cannot be ignored.

Bactericidal and virucidal activity of the alkalophilic P395D/L241V/T343A mutant of vanadium chloroperoxidase.

AIMS: Vanadium chloroperoxidase and its directed evolution mutant P395D/L241V/T343A were investigated for their antibacterial and antiviral potential at slightly alkaline pH and at a H(2)O(2) concentration that is low compared to current nonenzymatic formulations. METHODS AND RESULTS: Two bacteria (the Gram-negative Pseudomonas aeruginosa and the Gram-positive Staphylococcus aureus) and two viruses (the enveloped Herpes Simplex Virus and the nonenveloped Coxsackievirus B4) were incubated with the P395D/L241V/T343A mutant, 10 mmol l(-1) H(2)O(2) and 100 mmol l(-1) Br(-) at pH 8. Strong microbial reduction was observed and bactericidal and virucidal activities of the mutant were three to six orders of magnitude higher than for the wild-type enzyme. CONCLUSIONS: The P395D/L241V/T343A mutant of vanadium chloroperoxidase has a broad antimicrobial activity at alkaline conditions. SIGNIFICANCE AND IMPACT OF THE STUDY: For many disinfection formulations, antimicrobial activity at slightly alkaline pH values is required. To date, only the wild-type vanadium chloroperoxidase has been studied for its antibacterial activity, and only at acidic to neutral pH values. Its antiviral activity (e.g. useful for the cleaning of medical equipment) was not studied before. The observed activity for the alkalophilic P395D/L241V/T343A mutant is an important step forward in the application of this robust enzyme as a component in disinfection formulations.

[Fruit of the emergence of an enterovirus: acute haemorrhagic conjunctivitis]. / Fruit de l'émergence d'un entérovirus : la conjonctivite aiguë hémorragique.

First seen in Ghana and Indonesia in the early 70's, acute haemorrhagic conjunctivitis or "Apollo 11" disease is an eye infection caused by Enterovirus type 70 (EV70). The disease appeared to be a highly contagious conjunctivitis which spread rapidly all over the world. EV70 has been considered as an emerging virus and was classified as a new Enterovirus. No human or animal virus genetically similar to EV70 was known before the sudden outcome of the disease in Ghana, West Africa. EV70 appeared as a pretty demonstrative example of virus emergence and virus spreading. Studies of virus genetic mutations emphasized the variations of RNA virus within a short time period. The current review presents the EV70 infection and the genetic profile of the virus from its emergence to nowadays.

[Relationship between enteroviruses and diabetes]. / Vers une meilleure compréhension de la relation entre entérovirus et diabète de type 1.

Environmental factors, especially viruses, are involved in the initiation or the acceleration of type 1 diabetes (T1D) pathogenesis. Epidemiological data strongly suggest that enteroviruses, like coxsackievirus B4 (CV-B4), can be associated with T1D. It has been demonstrated that enterovirus infections were significantly more prevalent in at risk individuals, such as siblings of diabetic patients, when they developed anti-b cells autoantibodies or T1D, and in recently diagnosed diabetic patients, compared with control subjects. The isolation of CV-B4 from the pancreas of diabetic patients strengthened the hypothesis of a relationship between the virus and the disease. Studies performed in vitro and in vivo in animal models helped in discovering mechanisms of the infection of pancreas and other tissues, able to play a role in the pathogenesis of T1D. Interestingly, it cannot be excluded that enteroviruses behave as half-devil half-angel since experimental studies suggest that, in certain conditions, these agents would be able to protect individuals against the disease.

Sanger sequencing versus INNO-LiPA® HBV PreCore assay for routine detection of precore and basal core promoter mutations in hepatitis virus B chronically infected patients.

We compared the Sanger sequencing and the commercial INNO-LiPA® HBV assay for the routine detection of precore (PC) and basal core promoter (BCP) mutations of hepatitis B virus in chronically infected patients. The overall agreement rate between assays was 94.2% and 98.8% for the detection of PC and BCP mutations, respectively.

Coxsackievirus-B4E2 can infect monocytes and macrophages in vitro and in vivo.

Viral RNA (vRNA) is found in mice inoculated with coxsackievirus-B4E2 (CV-B4E2). The CV-B4E2 infection of murine spleen cells in vitro is enhanced with CV-B4E2-infected mouse serum. It has been investigated whether monocyte/macrophages were targets of CV-B4E2 in mice. vRNA has been detected in spleen and bone marrow of infected animals. The levels of vRNA were higher in CD14+ cells than in CD14- spleen cells and in F4/80- cells than in F4/80+ spleen cells. Meanwhile, CD14+ cells and F4/80- cells were more permissive to CV-B4E2 in vitro and the infection was enhanced when the virus was mixed with immune serum. While CV-B4E2 infected BMDM cultures (98% F4/80+); however, the immune serum did not enhance the infection. In conclusion, CV-B4E2 infects monocytes (CD14+, F4/80-) and macrophages (CD14+, F4/80+) in vivo and immune serum can enhance the in vitro infection of these cells arising out of the spleen.

[Influenza A/H5N1 virus outbreaks and prepardness to avert flu pandemic]. / Menace liée au virus influenza A/H5N1 et mise en place de la prévention face à un risque de pandémie de grippe.

This review emphasizes the need to improve the knowledge of the biology of H5N1 virus, a candidate for causing the next influenza pandemic. In-depth knowledge of mode of infection, mechanisms of pathogenesis and immune response will help in devising an efficient and practical control strategy against this flu virus. We have discussed limitations of currently available vaccines and proposed novel approaches for making better vaccines against H5N1 influenza virus. They include cell-culture system, reverse genetics, adjuvant development. Our review has also underscored the concept of therapeutic vaccine (anti-disease vaccine), which is aimed at diminishing 'cytokine storm' seen in acute respiratory distress syndrome and/or hemophagocytosis.

[Role of N-linked glycans in the functions of hepatitis C virus envelope glycoproteins]. / Rôle des N-glycanes dans les fonctions des glycoprotéines d'enveloppe du virus de l'hépatite C.

Hepatitis C virus (HCV) is an enveloped virus and encodes two envelope glycoproteins, E1 and E2. E1 and E2 are transmembrane type I proteins with a N-terminal ectodomain and C-terminal anchor. During their synthesis, E1 and E2 ectodomains are targeted in the endoplasmic reticulum lumen where they are modified by N-linked glycosylation. After their synthesis, E1 and E2 assemble as a non-covalent heterodimer. The N-linked glycosylation is based on the recognition of specific asparagine residue in the context of the consensus sequence Asn-X-Ser/Thr. E1 contains potentially 4 or 5 N-linked glycosylation sites and E2 up to 11. Recent data indicated that some glycans of glycoproteins E1 and E2 play a major role in protein folding and heterodimer formation. Some N-linked glycans of E2 were involved in interactions with CD81, a putative cellular receptor for HCV. It appeared that N-linked glycans of E1 and E2 played an important role of in the viral entry.