Rachel Desailloud

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.

Detection of enterovirus RNA in postoperative thyroid tissue specimens

Context  Autoimmune thyroiditis is a very common disease. A genetic predisposition and environmental factors such as viruses are thought to contribute to the development of autoimmune thyroiditis. Enteroviruses, which are involved in other autoimmune diseases, are attractive candidates.

Immunohistochemical- and PCR-based assay for the reproducible, routine detection of erythrovirus B19 in thyroid tissues

It is generally accepted that thyroid follicle cells are at least semi‐permissive for erythrovirus B19 (EVB19). Thus, various laboratory techniques have been successfully used to detect EVB19 in the thyroid gland, including polymerase chain reaction (PCR), immunohistochemistry, and in situ hybridization. However, the detection of EVB19 within the thyroid gland is problematic, and none of the detection protocols in the literature have been unequivocally validated. This multidisciplinary study in which 32 thyroidectomy subjects undergoing thyroidectomy in a French University hospital were prospectively recruited was performed over a period of 3 years. Prior to surgery, all the subjects were assayed for blood levels of anti‐EVB19 antibodies and (using a quantitative PCR [qPCR] assay) EVB19 itself. A qPCR assay for EVB19 and an immunohistochemical assay (based on polyclonal anti‐VP2 antibodies) were performed on the thyroidectomy samples. None of the subjects had an acute EVB19 infection. A viral load was detected in two serum samples and six thyroid biopsies. Three subjects had both a positive immunohistochemical assay and a positive qPCR assay for the thyroid tissue. It is noteworthy that the thyroid immunohistochemical and qPCR assays were negative in the two patients with detectable serum loads of EVB19. In conclusion, EVB19 can be detected in thyroid follicle cells by using immunohistochemical and qPCR assays. Ideally, patients should be tested with both PCR and immunohistochemical assays, in order to unequivocally confirm or rule out the presence of EVB19 in the thyroid gland. The present protocol must now be validated in larger series — notably with respect to its reliability and in order to determine qPCR positivity thresholds for application in future large‐scale studies. J. Med. Virol. 87:1054–1059, 2015.

Viruses and Type 1 Diabetes: Focus on the Enteroviruses

Type 1 diabetes (T1D) is one of the most common chronic diseases in developed countries and represents about 10% of all cases of diabetes. It is caused by a selective destruction of insulin-producing β cells in the pancreas. The disease has two subtypes: 1A, which includes the common, immune-mediated forms of the disease; and 1B, which includes nonimmune forms. In this review, we focus on subtype 1A, which for simplicity will be referred to as type 1 diabetes. [81, 34]. An increasing incidence rate of T1D has been observed for the last few decades especially in young individuals (less than five years old) [163]. The cause of T1D is still unknown. Several factors interact and lead to the development of the disease. An inflammatory islet infiltrate (insulitis) can be observed at the symptomatic onset of T1D, and reflects the immune response to β-cells [45]. T1D is an autoimmune disease, which implies a role of immune response effectors in the pathogenic processes and a failure of tolerance to‐ wards β-cell antigens.The susceptibility to T1D is influenced by genetic factors. More than 20 loci in addition to those located in the human leukocyte antigen (HLA) class II locus (es‐ pecially DQ and DR) on chromosome 6 are involved. Another contribution to the pathogen‐ esis of the disease could rely on epigenetic modifications (such as DNA methylation) and parent-of-origin effects [11]. Genetic modifications in the population cannot explain the rap‐ idly increased incidence of T1D in most populations. Altogether, the incidence variation from one season to another, the relationship between immigration and disease develop‐ ment, and the differences in incidence in different parts of the world in neighboring popula‐ tions with similar genetic profiles, suggest that the disease is a result of interaction of genetic and environmental factors [94].

Antibodies to VP1 of swine pasivirus in humans without evidence of transmission from a pig source

BACKGROUND Swine pasivirus (SPaV1) is a recently described enteric virus close to human parechoviruses and highly prevalent in pigs. Antibodies to Escherichia coli-expressed VP1 of SpaV1 have been found in a majority of humans in China. OBJECTIVES The objectives were to estimate the antibody prevalence in a European country, to test if exposure to the virus was linked to pig products and if this exposure was a risk factor for the development of diabetes type 1. STUDY DESIGN An ELISA test was developed and used to screen 842 healthy subjects with known exposure to pig products, 39 patients with diabetes type 1 and 20 controls. RESULTS We identified a high seroprevalence (15.6%) reacting to VP1 of SPaV1 among healthy human subjects. Analysis of risk factors argues against cross-species transmission from pigs as the source of infection. Data also indicate that the presence of SPaV1 VP1-binding antibodies is not associated with diabetes type 1 in humans. CONCLUSION Our results suggest that the seroreactivity frequently found in humans against SpaV1 is due to cross-reactivity with related antigen, perhaps a picornavirus, and that SpaV1 is not a zoonotic virus.

Antibody-Dependent Enhancement of Coxsackievirus-B Infection: Role in the Pathogenesis of Type 1 Diabetes

Antibodies can prevent viruses from infecting target cells, but antibodies against viruses can also enhance the infection of target cells. This phenomenon is called antibody-dependent enhancement (ADE) of infection. The mechanisms of ADE of infection and the results of this phenomenon in vivo are discussed. The ADE of CVB infection has been observed in animal models. In the human system it has been shown in vitro that CVB4, in combination with non-neutralizing antibodies, can infect human monocytes and stimulate the production of IFN-a by these cells through interactions with a specific viral receptor (CAR) and receptors for the Fc portion of IgG (FcgRII and FcgRIII). It cannot be excluded that the ADE of CVB infection in humans can increase the infection of peripheral blood mononuclear cells by these viruses, can cause viral escape from the immune response, and may contribute to the spread of CVBs in the host. Therefore, antibodies enhancing CVB infection may play a role in the pathogenesis of type 1 diabetes induced by or associated with these viruses.

Fluoxetine can inhibit coxsackievirus-B4 E2 in vitro and in vivo

Group B Coxsackieviruses (CV-B) are responsible for various acute human diseases, and they are involved in chronic diseases such as type 1 diabetes. It has been reported that fluoxetine (FLX) inhibited CV-B4E2 in human cell lines in vitro. In so far as CV-B4E2 can replicate in CD1 mice, it was investigated whether FLX could inhibit CV-B4E2 in vitro and in vivo in mouse systems. When 5.5 μM FLX was added to CV-B4E2-infected Min-6 cell (murine pancreas beta cell line) cultures, the virus-induced cytopathic effect was inhibited. In this system and in CV-B4E2-infected CD1 mouse pancreatic organotypic cultures treated with FLX the levels of infectious particles in supernatant fluids were below the limit of detection of the assay. The administration of FLX (10 mg/kg/day) by intraperitoneal route resulted in significant reduced levels of infectious particles in heart and pancreas of mice inoculated with CV-B4E2 by the same route. In conclusion FLX can inhibit CV-B4 in vitro and in vivo in mouse systems, additional studies are needed to investigate further the potential value of FLX to combat CV-B4 infections and to treat CV-B4-induced diseases.

Exposure of Piglets to Enteroviruses Investigated by an Immunoassay Based on the EV-G1 VP4 Peptide

Objective: The aim of this study was to investigate the exposure of piglets to enteroviruses-G (EV-G) through the presence of antibodies in their serum. Methods: Serum samples were obtained from the vena cava of 10 piglets at 9 weeks of age and again 39 days later (day 39). They were tested using an immunoassay based on the EV-G1 VP4 peptide, since VP4 is highly conserved among the four Enterovirus capsid proteins, and by using a seroneutralization assay. Results: For each serum collected on day 39 the optical density was high compared to the value obtained in serum collected earlier (p = 0.002). However, the titers of anti-EV-G1 serum neutralizing activity were not different in paired samples (p > 0.999). The sequence alignment of the EV-G1 VP4 peptide, encompassing 50 amino acids, used in the immunoassay showed 88% homology with EV-G, suggesting that antibodies directed toward other EV-G than EV-G1 may be detected. Conclusion: An immunoassay based on EV-G1 VP4 can detect an increased level of EV-G antibodies in piglet serum samples. Further studies are needed to determine whether this immunoassay may be useful for diagnosis and/or epidemiological studies and to monitor EV-G infection in pigs to evaluate strategies aimed to prevent enterovirus infections.