Activation of Nod2 signaling upon norovirus infection enhances antiviral immunity and susceptibility to colitis.

Over 90% of epidemic non-bacterial gastroenteritis are caused by human noroviruses (NoVs), which persist in a substantial subset of people allowing their spread worldwide. This has led to a significant number of endemic cases and up to 70,000 children deaths in developing countries. NoVs are primarily transmitted through the fecal-oral route. To date, studies have focused on the influence of the gut microbiota on enteric viral clearance by mucosal immunity. In this study, the use of mouse norovirus S99 (MNoV_S99) and CR6 (MNoV_CR6), two persistent strains, allowed us to provide evidence that the norovirus-induced exacerbation of colitis severity relied on bacterial sensing by nucleotide-binding oligomerization domain 2 (Nod2). Consequently, Nod2-deficient mice showed reduced levels of gravity of Dextran sodium sulfate (DSS)-induced colitis with both viral strains. And MNoV_CR6 viremia was heightened in Nod2-/- mice in comparison with animals hypomorphic for Atg16l1, which are prone to aggravated inflammation under DSS. Accordingly, the infection of macrophages derived from WT mice promoted the phosphorylation of Signal Transducer and Activator of Transcription 1 (STAT1) and NOD2's expression levels. Higher secretion of Tumor Necrosis Factor alpha (TNFα) following NOD2 activation and better viral clearance were measured in these cells. By contrast, reduced levels of pSTAT1 and blunted downstream secretion of TNFα were found in Nod2-deficient macrophages infected by MNoV_S99. Hence, our results uncover a previously unidentified virus-host-bacterial interplay that may represent a novel therapeutic target for treating noroviral origin gastroenteritis that may be linked with susceptibility to several common illnesses such as Crohn's disease.

Aberrant anti-viral response of natural killer cells in severe asthma.

Rhinovirus infections are the main cause of asthma exacerbations. As natural killer (NK) cells are important actors of the antiviral innate response, we aimed at evaluating the functions of NK cells from severe asthma patients in response to rhinovirus-like molecules or rhinoviruses.Peripheral blood mononuclear cells from patients with severe asthma and healthy donors were stimulated with pathogen-like molecules or with the rhinoviruses (RV)-A9 and RV-2. NK cell activation, degranulation and interferon (IFN)-γ expression were analysed.NK cells from severe asthma patients were less cytotoxic than those from healthy donors in response to toll-like receptor (TLR)3, TLR7/8 or RV-A9 but not in response to RV-2 stimulation. Furthermore, when cultured with interleukin (IL)-12+IL-15, cytokines which are produced during viral infections, NK cells from patients with severe asthma were less cytotoxic and expressed less IFN-γ than NK cells from healthy donors. NK cells from severe asthmatics exhibited an exhausted phenotype, with an increased expression of the checkpoint molecule Tim-3.Together, our findings indicate that the activation of NK cells from patients with severe asthma may be insufficient during some but not all respiratory infections. The exhausted phenotype may participate in NK cell impairment and aggravation of viral-induced asthma exacerbation in these patients.

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.

Protective role of murine norovirus against Pseudomonas aeruginosa acute pneumonia.

The murine norovirus (MNV) is a recently discovered mouse pathogen, representing the most common contaminant in laboratory mouse colonies. Nevertheless, the effects of MNV infection on biomedical research are still unclear. We tested the hypothesis that MNV infection could alter immune response in mice with acute lung infection. Here we report that co-infection with MNV increases survival of mice with Pseudomonas aeruginosa acute lung injury and decreases in vivo production of pro-inflammatory cytokines. Our results suggest that MNV infection can deeply modify the parameters studied in conventional models of infection and lead to false conclusions in experimental models.

Survival of Enveloped and Non-Enveloped Viruses on Inanimate Surfaces.

In the present study, we evaluated the viability of non-enveloped viruses, minute virus of mice (MVM) and coxsackievirus B4 (CVB4), and enveloped-viruses, influenza A virus (H1N1) and herpes simplex virus type 1 (HSV-1), on surfaces. We also investigated the impact of the initial concentration of proteins and sodium chloride on the persistence of infectious CVB4 on surfaces. Viral suspensions (>10(4.5) TCID50) were applied to petri dish lids and dried under the air flow of a biosafety cabinet. The recovered viral preparations were titered on appropriate cell lines. Enveloped viruses persisted for less than 5 days while CVB4 and MVM persisted for weeks. However, repetitive cycles of drying and resuspension had a stronger virucidal effect on CVB4 than on H1N1 and HSV-1. These repetitive cycles had no effect on the infectious titer of MVM. When exposed to drying, the initial concentrations of bovine serum albumin (from 0 to 90 mg mL(-1)), fetal calf serum (from 0 to 100%), and sodium chloride (from 0 to 300 mg mL(-1)) affected the viability of CVB4. CVB4 was more likely to be inactivated by drying in a protein-rich medium, whereas the impact of drying was reduced in the presence of sodium chloride. The results of the present study demonstrated that the resistance of viruses to drying, as suggested by iterative drying, was not due to the heterogeneity of viral subpopulations, but was influenced by media compositions and component concentrations, as illustrated in the model of CVB4.

Diagnosis of viral infections using myxovirus resistance protein A (MxA).

BACKGROUND: Myxoma resistance protein 1 (MxA) is induced during viral infections. MxA testing could be helpful to differentiate between viral and bacterial infections. METHODS: A prospective multicenter cohort study was performed in pediatric emergency departments. MxA blood values were measured in children with confirmed viral or bacterial infections, uninfected controls, and infections of unknown origin. First patients were used to determine MxA threshold for viral infection. The diagnostic performance of MxA was determined by using receiver operating characteristic (ROC) analysis. Sensitivities (Se), specificities (Sp), and positive and negative likelihood ratios (LR+, LR-) were calculated. RESULTS: The study included 553 children; 44 uninfected controls and 77 confirmed viral infections (mainly respiratory syncytial virus and rotavirus) were used to determine an MxA threshold at 200 ng/mL. In the 193 other patients with confirmed infections and uninfected controls (validation group), MxA was significantly higher in patients with viral than in those with bacterial infections and uninfected controls (P < .0001). The area under the ROC curve (AUC) were 0.98, with 96.4% Se and 85.4% Sp, for differentiating uninfected from virus-infected patients and 0.89, with 96.4% Se and 66.7% Sp, for differentiating bacterial and viral infections. MxA levels were significantly higher in patients with clinically diagnosed viral versus clinically diagnosed bacterial infections (P < .001). Some patients with Streptococcus pneumonia infections had high MxA levels. Additional studies are required to elucidate whether this was due to undiagnosed viral coinfections. CONCLUSIONS: MxA is viral infection marker in children, at least with RSV and rotavirus. MxA could improve the management of children with signs of infection.

Viruses contained in droplets applied on warmed surface are rapidly inactivated.

Heat inactivation of viruses was reported, however, the thermal resistance of viruses in droplets has not been studied. The aim of this study was to evaluate the pattern of heat resistance of minute virus of mice (MVM), coxsackievirus B4 (CVB4), influenza A virus (H1N1), and herpes simplex virus type 1 (HSV1) contained in droplets. Four µL droplets containing viruses (> 10(4.5) TCID50) were applied onto warmed surface obtained by using a self-made heating device. Viral suspensions were exposed to temperatures ranging from 70 to 130°C for 0 to 90 min depending on the virus, and then the recovered viral preparations were tittered. Inactivation rates were calculated from curves that were analysed according to the first order kinetics model. Full inactivation was obtained for MVM in 90 min at 80°C and in 2 s at 130°C, for H1N1 in 14 s at 70°C and in 1 s at 110°C, for CVB4 and HSV-1 in 5 s and 7 s respectively at 70°C and in 1 s at 100°C. Clearly, MVM was more resistant than H1N1 that was more resistant than HSV-1 and CVB4, which was reflected by increasing inactivation rates. The impact of short time exposure to heat onto the infectivity of viruses contained in a small volume of suspension has been determined. For the first time, the inactivation of viral particles contained in drops exposed to temperatures higher than 100°C has been investigated. It appears that heating can have an unexpected faster virucidal effect than previously described.

Inactivation of coxsackievirus B4, feline calicivirus and herpes simplex virus type 1: unexpected virucidal effect of a disinfectant on a non-enveloped virus applied onto a surface.

OBJECTIVE: To evaluate the effect of a disinfectant onto viruses in suspension on the one hand and applied onto a surface on the other. METHODS: A system combining flocked swabs to recover viruses dried onto stainless steel carriers and gel filtration to eliminate cytotoxic products has been developed to study the virucidal effect of a quaternary ammonium-based disinfectant towards herpes simplex virus type 1 (HSV-1), coxsackievirus B4 (CVB4) and feline calicivirus F9 (FCV). The recovery of FCV has been estimated by RT real-time PCR. RESULTS: HSV-1, CVB4 and FCV had a titer over 10(4) TCID50 · ml(-1) after 2 h drying and were recovered from the carriers using flocked swabs. HSV-1 was inactivated in suspension and on stainless steel carriers by the disinfectant (a reduction factor of 4 and 2.83 log, respectively) whereas CVB4 was resistant. The reduction of infectious titer was moderate, 1.5 log in 30 min, when FCV was in suspension, whereas it was up to 4 log in 10 min when the virus was dried on a carrier. Dried FCV was efficiently recovered from carriers as demonstrated by RT real-time PCR. CONCLUSION: A non-enveloped virus, FCV, applied on a surface, but not in suspension, was inactivated by a quaternary ammonium-based disinfectant. The resistance of viruses applied onto a surface to the effect of disinfectants should be investigated further.

Inactivation of an enterovirus by airborne disinfectants.

BACKGROUND: The activity of airborne disinfectants on bacteria, fungi and spores has been reported. However, the issue of the virucidal effect of disinfectants spread by fogging has not been studied thoroughly. METHODS: A procedure has been developed to determine the virucidal activity of peracetic acid-based airborne disinfectants on a resistant non-enveloped virus poliovirus type 1. This virus was laid on a stainless carrier. The products were spread into the room by hot fogging at 55°C for 30 minutes at a concentration of 7.5 mL.m(-3). Poliovirus inoculum, supplemented with 5%, heat inactivated non fat dry organic milk, were applied into the middle of the stainless steel disc and were dried under the air flow of a class II biological safety cabinet at room temperature. The Viral preparations were recovered by using flocked swabs and were titered on Vero cells using the classical Spearman-Kärber CPE reading method, the results were expressed as TCID50.ml(-1). RESULTS: The infectious titer of dried poliovirus inocula was kept at 10(5) TCID50.mL(-1) up to 150 minutes at room temperature. Dried inocula exposed to airborne peracetic acid containing disinfectants were recovered at 60 and 120 minutes post-exposition and suspended in culture medium again. The cytotoxicity of disinfectant containing medium was eliminated through gel filtration columns. A 4 log reduction of infectious titer of dried poliovirus inocula exposed to peracetic-based airborne disinfectant was obtained. CONCLUSION: This study demonstrates that the virucidal activity of airborne disinfectants can be tested on dried poliovirus.

Persistent infection of thymic epithelial cells with coxsackievirus B4 results in decreased expression of type 2 insulin-like growth factor.

It has been hypothesized that a disturbance of central self-tolerance to islet ß cells may play a role in the enteroviral pathogenesis of type 1 diabetes. Whether enteroviruses can induce an impaired expression of ß-cell self-antigens in thymic epithelial cells has been investigated in a murine thymic epithelial (MTE) cell line. This cell line was permissive to the diabetogenic group B4 coxsackievirus (CV-B4) strain CV-B4 E2 and spontaneously expressed type 2 insulin-like growth factor (Igf2), the dominant self-antigen of the insulin family. In this model, a persistent replication of CV-B4 E2 was obtained, as attested to by the prolonged detection of intracellular positive- and negative-strand viral RNA by reverse transcription-PCR (RT-PCR) and capsid protein VP1 by immunofluorescent staining and by the release of infectious particles in culture supernatants. The chronic stage of the infection was characterized by a low proportion of VP1-positive cells (1 to 2%), whereas many cells harbored enteroviral RNA, as displayed by RT-PCR without extraction applied directly to a few cells. Igf2 mRNA and IGF-2 protein were dramatically decreased in CV-B4 E2-infected MTE cell cultures compared with mock-infected cultures, whereas housekeeping and interleukin-6 (Il6) gene expression was maintained and Igf1 mRNA was decreased, but to a lower extent. Inoculation of CV-B3, CV-B4 JVB, or echovirus 1 resulted in a low level of IGF-2 in culture supernatants as well, whereas herpes simplex virus 1 stimulated the production of the protein. Thus, a persistent infection of a thymic epithelial cell line with enteroviruses like CV-B4 E2 can result in a disturbed production of IGF-2, a protein involved in central self-tolerance toward islet ß cells.

Infection of primary cultures of murine splenic and thymic cells with coxsackievirus B4.

Infection of primary cultures of total splenic and thymic cells from BALB/c and C3H/HeN mice with CVB4 E2 and JVB strains has been investigated. The presence of positive-strand viral RNA within cells was determined by semi-nested RT-PCR, and viral replication was attested by detection of intracellular negative-strand viral RNA and by release of infectious particles in culture supernatants. Viral replication occurred with both CVB4 strains to an extent dependent on the genetic background of the host. No interferon-alpha production was detected in the supernatants of CVB4-infected cultures using biological titration. Together these results suggest that infection of splenic and thymic cells can play a role in virus dissemination, and therefore in the pathophysiology of CVB4 infections.

A part of the VP4 capsid protein exhibited by coxsackievirus B4 E2 is the target of antibodies contained in plasma from patients with type 1 diabetes.

The capsid protein VP4 was identified previously as the target of antibodies contained in plasma enhancing the coxscakievirus B4 (CV-B4) E2-induced production of IFN-alpha by peripheral blood mononuclear cells (PBMCs). The sequence of VP4 recognized by these antibodies was investigated. This sequence was identified as amino acids 11 to 30 by using synthetic overlapping peptides spanning VP4(CV-B4 E2) in competition experiments for antibodies enhancing the CV(B4 E2) induced production of IFN-alpha by PBMCs. This amino acid sequence was the major target of anti-VP4 antibodies according to enzyme-linked immunosorbent assays (ELISA). There was a positive correlation between the levels of anti-VP4 and anti-VP4(11-30) peptide antibodies detected by ELISA. The levels and the prevalences of these antibodies were significantly higher in patients with type 1 diabetes than in healthy controls. The proportions and the levels of those antibodies in patients were independent of HLA-DR alleles, age, or presence of ketosis in blood and were not associated with newly or previously diagnosed disease. The VP4(CV-B4 E2) amino acid sequence was submitted to the Swiss-model in project mode to visualize the possible shape of the sequence of VP4 corresponding to amino acids 11-30 which appeared to be constituted principally by an non-structured loop. In conclusion, the sequence of VP4 corresponding to amino acids 11-30, or a part of it plays a role in the plasma-dependent enhancement of CV-B4 E2-induced production of IFN-alpha by PBMCs, suggesting that at 37 degrees C the virus exhibits that region of VP4 to antibodies.

Role of the capsid protein VP4 in the plasma-dependent enhancement of the Coxsackievirus B4E2-infection of human peripheral blood cells.

It has been previously shown that antibodies contained in human plasma directed towards the Coxsackievirus B4 (CVB4)E2 capsid protein VP4 can enhance the CVB4E2-induced production of IFN-alpha by peripheral blood mononuclear cells (PBMC). The aim of this study was to produce a VP4 fusion protein to investigate the role of the internal capsid protein VP4 and anti-VP4 antibodies in the plasma-dependent enhancement of CVB4E2 infection of PBMC. A fusion protein MBPVP4 containing the VP4 insert of CVB4E2 and a control fusion protein MBP-beta-gal-alpha, were produced in Escherichia coli K12 TB1. The CVB4E2 infection of PBMC was quantified by using a real time PCR method amplifying CVB4E2-negative strand RNA. IFN-alpha concentrations in culture supernatants were assayed by DELFIA. MBPVP4 but not MBP-beta-gal-alpha, preincubated with plasma inhibited the plasma-dependent enhancement of CVB4E2-induced production of IFN-alpha by PBMC. Human plasma samples, antibodies contained in plasma eluted from MBPVP4-coated plates, but not from MBP-beta-gal-alpha-coated plates, incubated with CVB4E2 enhanced the infection of PBMC and the production of IFN-alpha by infected cells. Together our results show that VP4 and anti-VP4 antibodies play a role in the plasma-dependent enhancement of CVB4E2 infection of PBMC.

Prolonged viral RNA detection in blood and lymphoid tissues from coxsackievirus B4 E2 orally-inoculated Swiss mice.

The spreading of viral RNA within Swiss Albino mice orally inoculated with coxsackievirus B4 E2 strain (CVB4 E2) was studied by using RT-PCR and semi-nested-RT-PCR methods. Viral RNA was detected in various organs: pancreas, heart, small intestine, spleen, thymus, and blood at various postinfectious (p.i.) times ranging from 8 hr to 150 days. Our results show that (i) outbred mice can be infected with CVB4 E2 following an oral inoculation, which results in systemic spreading of viral RNA, (ii) CVB4 E2 infection can be associated with a prolonged detection of viral RNA in spleen, thymus and blood, up to 70 days p.i. and further in other organ tissues.

Correlation between the anti-virus-induced cytopathic effect activity of interferon-alpha subtypes and induction of MxA protein in vitro.

There are several interferon-alpha (IFN-alpha) subtypes. Mechanism of disparity in biological effects among members of IFN-alpha subtypes remains unexplained. Biological activity of IFN-alpha is mediated in part by induction of intracellular antiviral proteins. We studied whether differences in biologic effects of IFN-alpha subtypes may rely on their antiviral protein inducing effect. Intracellular induction of MxA protein and anti-virus-induced cytopathic effect (CPE) activity of 11 IFN-alpha subtypes in human amnion WISH cells have been studied. MxA protein quantitation in cell lysates was performed by immunochemiluminescence assay and anti-virus-induced CPE activity was assessed by protection against vesicular stomatitis virus (VSV)-induced CPE. Range of MxA values was high when cells were treated with 10 and 100 IU/ml of each IFN-alpha subtype. Levels of MxA correlated with anti-VSV-induced CPE obtained with 10 IU/ml IFN-alpha subtype. Together our data show a disparity in MxA-inducing activity of IFN-alpha subtypes and suggest that differences in anti-VSV-induced CPE of IFN-alpha subtypes in WISH cells can be related to their different ability to induce MxA.

Viral protein VP4 is a target of human antibodies enhancing coxsackievirus B4- and B3-induced synthesis of alpha interferon.

Coxsackievirus B4 (CVB4)-induced production of alpha interferon (IFN-alpha) by peripheral blood mononuclear cells (PBMC) is enhanced in vitro by nonneutralizing anti-CVB4 antibodies from healthy subjects and, to a higher extent, from patients with insulin-dependent diabetes mellitus. In this study, we focused on identification of the viral target of these antibodies in CVB systems. High levels of IFN-alpha were obtained in supernatants of PBMC incubated with CVB4E2 or CVB3 and plasma from healthy subjects and, to a higher extent, from patients. The VP4 capsid proteins dissociated by heating at 56 degrees C from CVB4E2 (VP4(CVB4)) and CVB3 (VP4(CVB3)) but not H antigen preincubated with plasma from healthy subjects or patients inhibited the plasma-dependent enhancement of CVB4E2- and CVB3-induced IFN-alpha synthesis. There was no cross-reaction between VP4(CVB4) and VP4(CVB3) in the inhibiting effect. IFN-alpha levels in culture supernatants showed dose-dependent correlation with anti-VP4 antibodies eluted from plasma specimens using VP4-coated plates. There were higher index values for anti-VP4 antibodies detected by enzyme-linked immunosorbent assay (ELISA) and higher proportions of positive detection in 40 patients than in 40 healthy subjects (80% versus 15% for anti-VP4(CVB4)). There was no relationship between the levels of anti-CVB neutralizing antibodies and the detection of anti-VP4 antibodies by ELISA. The CVB plasma-induced IFN-alpha levels obtained in PBMC cultures in the anti-VP4 antibody-positive groups were significantly higher than those obtained in the anti-VP4 antibody-negative groups regardless of the titers of anti-CVB neutralizing antibodies. These results show that VP4 is the target of antibodies involved in the plasma-dependent enhancement of CVB4E2- and CVB3-induced IFN-alpha synthesis by PBMC.

DNA microarray to monitor the expression of MAGE-A genes.

BACKGROUND: The MAGE-A genes encode antigens that are of particular interest for antitumor immunotherapy because they are strictly tumor specific and are shared by many tumors. We developed a rapid method to identify the MAGE-A genes expressed in tumors. METHODS: A low-density DNA microarray was designed to discriminate between the 12 MAGE-A cDNAs amplified by PCR with only one pair of consensus primers. The assay involved reverse transcription of total RNA with oligo(dT) primer, followed by PCR amplification and hybridization on a microarray. Amplification in the presence of Biotin-16-dUTP allowed subsequent detection of the amplicons on the microarray carrying 12 capture probes, each being specific for a MAGE-A gene. Probe-amplicon hybrids were detected by a streptavidin-based method. RESULTS: PCR conditions were optimized for low detection limits and comparable amplification efficiencies among all MAGE-A nucleotide sequences. The microarray assay was validated with a panel of 32 samples, by comparison with well-established reverse transcription-PCR assays relying on amplification with primers specific for each gene. Virtually identical results were obtained with both methods, except for MAGE-A3 and MAGE-A5. Detection of MAGE-A5 was more sensitive with the microarray assay. Detection of MAGE-A3 was hampered by the presence of MAGE-A6, which is 98% identical: the MAGE-A3 capture probe cross-hybridized with MAGE-A6 amplicons because these sequences differed by only a single base. CONCLUSIONS: This post-PCR microarray assay could be useful to evaluate MAGE expression in tumors before therapeutic vaccinations with MAGE-A gene products.