Delphine Caloone

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‐α 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 VP4CV‐B4 E2 in competition experiments for antibodies enhancing the CVB4 E2 induced production of IFN‐α 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‐VP411–30 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 VP4CV‐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‐α by PBMCs, suggesting that at 37°C the virus exhibits that region of VP4 to antibodies. J. Med. Virol. 80:866–878, 2008.

Persistent Infection of Thymic Epithelial Cells with Coxsackievirus B4 Results in Decreased Expression of Type 2 Insulin-Like Growth Factor

ABSTRACT It has been hypothesized that a disturbance of central self-tolerance to islet β cells may play a role in the enteroviral pathogenesis of type 1 diabetes. Whether enteroviruses can induce an impaired expression of β-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 β 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‐α 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.

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.

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 (>104.5 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.

Persistent infection of a carcinoma thyroid cell line with coxsackievirus B.

BACKGROUND Viral infections are described as environmental factors that are implicated in various thyroid diseases. The role of enteroviruses (EV) in the pathogenesis of thyroid diseases has been suspected. Recently, we found that EV RNA could be detected in postoperative thyroid specimens. We decided to investigate the infection of a human thyroid cell line with coxsackievirus B4 (CVB4). METHODS The wild-type human thyroid carcinoma cells K1 were inoculated with CVB4E2 at 2.1 x 10(7) TCID50/mL. The titer of the virus progeny was determined every 3 days on HEp-2 cells. CVB replication at the molecular level was monitored by searching for intracellular viral genomes using reverse transcription (RT)-polymerase chain reaction (PCR). EV VP1 capsid protein was detected by indirect immunocytofluorescence staining. Cell viability was determined by 3-(4,5)-dimethylthiahiazo(-z-y1)-3,5-di-phenytetrazoliumromide (MTT) absorbance, and the nuclear morphology was assessed by Hoechst Dye staining. RESULTS Infectivity assays with CVB4E2 revealed an increase in viral titers. Virus production decreased thereafter, but was not stopped by serial subculture for 24 days after infection. Detection of intracellular positive and negative viral RNA strands by RT-PCR was positive between days 1 and 14 postinfection (p.i.), and by semi-nested RT-PCR up to 24 days. K1 cell cultures infected with CVB4 were stained positively for EV VP1: the number of VP1-positive cells decreased rapidly within 6 days and remained low up to the end of culture. Compared with mock-infected cultures, viability in CVB4-infected cultures was around 100% up to 24 days. Cells with strongly fluorescent nuclei and/or fragmented nuclei were observed. CONCLUSION We demonstrate for the first time that CVB4 could replicate in thyroid cells and could persist, with predominance of viral negative RNA strands for up to 24 days p.i. without obvious cytopathic effect. Our results suggest that CVB4 may lead to thyroid cell apoptosis. Further studies are needed to determine whether CVB could play a role in thyroid pathologies.

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.

Prolonged viral RNA detection in the central nervous system of one-week-old Swiss albino mice following coxsackievirus B4 and echovirus 9 infection.

Objectives: Type B coxsackieviruses (CV-B), together with echoviruses (E), are among the most common pathogens encountered in aseptic meningitis and meningoencephalitis. They frequently infect the central nervous system (CNS). The mechanisms of virus spreading in the CNS are poorly understood. In the present study, we investigated CV-B4 and E-9 spreading and neurotropism within intraperitoneally inoculated one-week-old Swiss albino mice. Methods: Seminested RT-PCR and virus isolation were used to assay viral distribution. Results: Viral RNA was present in various organs: brain, spinal cord, spleen and heart at various times post-infection (p.i.); ranging from 1 day p.i. up to 30, 60 and 90 days p.i, respectively, for CV-B4-JVB-, E-9 Barty- and CV-B4-E2-infected mice. Organs became negative for virus isolation after 5 days p.i., except for brain and heart from CV-B4 E2-infected mice, which remained positive for up to 10 and 15 days p.i., respectively. Negative viral RNA strand was detected mainly in brain and spinal cord of infected mice until 30 and 60 days p.i. Conclusion: This is the first report on the persistence of CV-B4 and E-9 in the CNS of intraperitoneally inoculated mice.

Viral induction of Zac1b through TLR3- and IRF3-dependent pathways

Zinc finger protein regulator of apoptosis and cell cycle arrest (Zac1) is a transcription factor able to induce apoptosis or cell cycle arrest through independent pathways. In spite of the important potential functions attributed to Zac1, little is known of its physiological regulation and biological function. We discovered that variant Zac1b was expressed in murine embryonic fibroblasts (MEFs) treated with polyriboinosinic polyribocytidylic acid [poly(I:C)], a synthetic double-stranded RNA. This regulation occurred mainly through Toll-Like Receptor 3 (TLR3)- and Interferon Regulatory Factor 3 (IRF3)-dependent pathways. As TLR3 and IRF3 are central activators of antiviral immunity, we hypothesized that Zac1 may be implicated in antiviral responses. In line with this notion, we observed that Zac1b was expressed in MEFs infected with Encephalomyocarditis virus (EMCV). We also observed that Zac1-deficient MEFs were less sensitive to EMCV-induced cell death than wild-type MEFs. However, Zac1 gene inactivation had no effect on the survival of mice infected with EMCV. In conclusion, this study describes for the first time a transcriptional regulation of Zac1b, induced by synthetic dsRNA and RNA viruses, the functional significance of which remains to be further investigated.

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 (> 104.5 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.