Kinetics and pattern of viral excretion in biological specimens of two MERS-CoV cases.

BACKGROUND: Middle East respiratory syndrome coronavirus (MERS-CoV) is an emerging coronavirus involved in severe acute respiratory distress syndrome (ARDS) and rapid renal failure. Hospital outbreak and nosocomial transmission were reported, however, several issues remain on the viral excretion course. OBJECTIVES: Describe the kinetics and pattern of viral excretion in two infected patients. STUDY DESIGN: After the initial diagnosis, blood, urine, rectal and respiratory samples were collected regularly, aliquoted and stored at -80°C. Real-time reverse transcriptase polymerase chain reaction assay targeted the UpE and Orf1a regions of the MERS-CoV genome. RESULTS: In patient 1, who died of refractory ARDS and renal failure, MERS-CoV RNA was detected in pharyngeal and tracheal swabs, as well blood samples and urine samples until the 30th day. Rectal swabs were negative. Patient 2 also developed multiple-organ failure, but survived, with persisting renal insufficiency (creatinine clearance at 30 mL/min) and persistent interstitial syndrome albeit weaned off mechanical ventilation and no longer requiring oxygen. Tracheal aspirations were positive until the 33rd day, while nasopharyngeal swabs were negative. All other biological samples were negative. DISCUSSION: Lower respiratory tract excretion of MERS-CoV could be observed for more than one month. The most severely ill patient presented an expression of the virus in blood and urine, consistent with a type-1 interferon mediated immunological response impaired in patient 1, but developed by patient 2. These results suggest that infection control precautions must be adequately evaluated in clinical wards and laboratories exposed to MERS-CoV.

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.

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.

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.

[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.

[Development of duplex real-time RT-PCR for detection of influenza virus A and B]. / Mise au point d'une technique de RT-PCR duplex en temps réel pour la détection des virus influenza A et B.

Influenza viruses continue to be accountable for winter outbreaks. The potential for developing complications is higher in certain risk groups, such as children, the elderly and individuals with chronic medical conditions. We have presented a duplex real-time RT-PCR for detection of influenza A and B. The sensibility of our method was estimated at 0.01TCID(50)/ml for detection of influenza A and 0.1TCID(50)/ml for influenza B. Any other viruses with respiratory tract tropism were detected with our method. After that, we tested our duplex RT-PCR on 119 samples (nasal aspirates and liquids of broncho-alveolar washes) collected in the CHRU of Lille between November 2005 and January 2006 from patients aged one month to 81 years. Conventional methods such as direct immunofluorescence (IF) assay and/or cell culture were applied on these samples. Four samples were positive for influenza A virus detection and, in particular, one liquid of broncho-alveolar wash for which the direct IF assay was negative. Thus, our method is adapted to diagnosis of flu infections. Nevertheless, our duplex RT-PCR will be tested during a flu outbreak.

[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.

[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.

[Emergent viruses: SARS-associate coronavirus and H5N1 influenza virus]. / Virus respiratoires émergents : virus du Sras et virus influenza A/H5N1 hautement pathogène.

Two viral agents with RNA genome are responsible for emerging illnesses: influenza virus A/H5N1 and Severe Acute Respiratory Syndrome virus (SARS). For the diagnosis of SARS virus infection, an epidemiological investigation is necessary to know whether the patient has been exposed to a risk in a country where the SARS virus is circulating or whether the patient had worked in a laboratory handling SARS virus. The detection of SARS virus is possible in various clinical samples (including urine) by viral culture or RT-PCR. The handling of those samples and RNA extraction must be performed in a BSL3 laboratory. The SARS virus RT-PCR is poorly sensitive, therefore the test should be performed on samples collected consecutively for several days. In front of a suspicion of A/H5N1, similar procedures are recommended. An epidemiologic investigation is necessary to specify whether the patient stayed in a country where A/H5N1 virus was circulating. Clinical samples needed for a specific diagnosis are: nasopharyngeal, throat-swab or fecal samples, cerebrospinal fluid and blood. The presence of A/H5N1 virus is confirmed by viral isolation or RNA detection by RT-PCR. RNA extraction must be performed in a BSL3 laboratory. For diagnosis of A/H5N1 virus infection, RT-PCR test amplifies specifically a fragment of H5 gene (Hemagglutinin). In french laboratories of medical virology, procedures are ready to diagnose the first case of A/H5N1 virus infection and cases of reemerging SARS virus infection.

[Various approaches of therapeutic vaccination for the treatment of HIV type 1 infection]. / Différentes approches de vaccination thérapeutique dans le traitement de l'infection par le VIH-1.

HIV-1 infection is a major pandemic situation. With the advent of highly active antiretroviral therapy (HAART), morbidity and mortality associated with HIV-1 infection have been dramatically reduced. However, HAART does not enable eradication of the virus. The efficacy of these new regimens is limited by problems over long-term use such as toxicity and resistance. Therapeutic vaccination is an alternative approach to HIV-1 infection. The main aim is to boost and reinforce virus-specific host immune responses. Several immunogens and schedules of immunization have been tested. In this review, various strategies designed for therapeutic vaccines for HIV-1 infection are presented.

[Mechanisms of imiquimod indirect antiviral activity]. / Mécanismes de l'activité antivirale indirecte de l'imiquimod.

The potential role of an immune response in HPV-related anogenital disorders had already been anticipated by clinicians. Indeed the lesions efflorescence and the relapsing HPV infection in HIV positive patients as well as the lack of recurrence in patients with spontaneous cure, provided relevant clues for a likely immune mechanism. At present time, the role of the immune system in the development of HPV-related anogenital disorders is well established : HPV induce a humoral and cell mediated immune response. This response is mainly exerted towards infected cells; it is also exerted at the systemic level, through antibodies synthesis, but this pathway remains a secondary one. Due to the limits of the present therapies (either purely destructive and characterized by the rate of recurrences, or antiviral, but difficult to use), it was necessary to find a new treatment type which enhances the local immune response, results in the disappearance of lesions and allows for a decrease in the risk of recurrences. The original mechanism of action of the first cell-mediated immune response modifier: imiquimod, for local use (Aldara 5 % cream) is an answer to this need. The first positive results observed in vitro and in animals were confirmed in patients with HPV anogenital warts in a double blind placebo-controlled study: imiquimod inhibits HPV replication and results in the condyloma regression. Its action is based on the combined activation of the natural local immunity, by stimulating interferon alpha; and of the acquired immunity, by stimulating a T-cell mediated immune response. Thus imiquimod appears to be an original antiviral compound, because it does not act directly on the virus itself.

[Does dopamine play a role in renal anomaly in sodium excretion, a marker for hereditary predisposition to arterial hypertension?]. / Anomalie rénale de l'excrétion sodée, un marqueur de la prédisposition héréditaire à l'hypertension artérielle, rôle de la dopamine?

Since Dahl's observation, a renal defect os sodium excretion is proposed as one of pathogenetic mechanism of hypertension (HTA). Our study has tried to verify this concept in 20 young normotensives with (n = 12) and without (n = 8) familial predisposition to HTA, allowing to test the genetic transmission of such potential renal abnormality of sodium balance. Each people was submitted to 3 different Na diet (20, 170 and 340 mM NaCl) each for 1 week. At each visit, blood pressure, vascular resistances, biological values were determined at rest (plasma renin activity, creatinine clearance, 24 hours before the test, catecholamines, aldosterone and ion urinary excretion). Then 1 liter of isotonic saline was perfused in 30 minutes with measures of blood pressure and 3 hours urinary dopamine and Na excretion. During the low and medium Na diets, but not during the high Na diet, the natriuresis and dopamine excretion were lower in the 3 hour urine collection in patients with a family history of HTA (p less than 0.02 and p less than 0.005, respectively). No other clinical or biological difference was noted between the 2 groups. Thus, genetic hypertensive predisposition seems to be characterized by a lower Na excretion during acute Na loading in normal or depleted Na diet, linked to an impaired urinary dopamine excretion. These findings suggest that the defect responsible for the susceptibility to sodium intake is at the kidney level. Some dopamine agonists would be of great therapeutical value in treating such patients when blood pressure begins to rise.