Wassim Chehadeh

Increased Level of Interferon-α in Blood of Patients with Insulin-Dependent Diabetes Mellitus: Relationship with Coxsackievirus B Infection

The activation of the interferon (IFN)-alpha system and its relationship with coxsackievirus B (CVB) infection has been analyzed in 56 patients with insulin-dependent diabetes mellitus (IDDM; 25 children and 31 adults). Elevated levels of IFN-alpha were found in plasma of 70% of patients (39/56), and a positive detection of IFN-alpha mRNA in blood cells by reverse transcriptase-polymerase chain reaction (RT-PCR) was observed in 75% of patients (42/56). Enterovirus (EV) RNA assayed by seminested RT-PCR was detected in the blood of 50% of IFN-alpha-positive patients but not in any IFN-alpha-negative patients. The results of genotype analysis of amplified EV RNA sequences (5 CVB2, 8 CVB3, and 8 CVB4) were concordant with the results of CVB-neutralization tests. The comparison between IFN-alpha, EV RNA, and serology suggested that the proportion of CVB infection associated with IFN-alpha positivity might be higher than is predicted from the investigation of EV RNA. Together, the results suggest that, in a majority of cases, a CVB infection is associated with clinical IDDM.

Persistent Infection of Human Pancreatic Islets by Coxsackievirus B Is Associated with Alpha Interferon Synthesis in β Cells

ABSTRACT The interactions of coxsackievirus B3 (CVB3), CVB4E2 (diabetogenic), and CVB4JBV (nondiabetogenic) strains with human pancreatic islets from eight adult brain-dead donors were investigated. Persistent replication of viruses in human islets was proved by detection of viral RNA by in situ hybridization, VP1 capsid protein by immunofluorescence (IF) staining, negative-strand viral RNA by reverse transcription-PCR in extracted RNA from islets, and release of infectious particles up to 30 days after infection without obvious cytolysis. By double IF staining, glucagon-containing α cells and insulin-containing β cells were shown to be susceptible to CVB. The persistence of CVB3 and CVB4 in islet cells was associated with the chronic synthesis of alpha interferon (IFN-α), as evidenced by the detection of IFN-α mRNA and immunoreactive IFN-α with antiviral activity. By double IF staining, IFN-α was detected in insulin-producing β cells only. Experiments with neutralizing anti-coxsackievirus and adenovirus receptor (CAR) antibodies provided evidence that CAR was expressed by α and β cells and that it played a role in the infection of these cells with CVB and the consecutive IFN-α expression in β cells. The viral replication and the expression of IFN-α in islets were not restricted to the CVB4E2 diabetogenic strain and did not depend on the genetic background of the host. The neutralization of endogenous IFN-α significantly enhanced the CVB replication in islet cells and resulted in rapid destruction of islets. Thus, human β cells can harbor a persistent CVB infection, and CVB-induced IFN-α plays a role in the initiation and/or maintenance of chronic CVB infection in human islets.

Persistent Infection of Human Thymic Epithelial Cells by Coxsackievirus B4

ABSTRACT Persistent replication of coxsackievirus B4 (CVB4) E2 (diabetogenic) and CVB4 JBV (nondiabetogenic) strains in thymic epithelial cell (TEC)-enriched cultures (≥95%) was proved by detection of positive- and negative-strand viral RNA by reverse transcription-PCR in extracted RNA from cell cultures, VP1 capsid protein detection by immunofluorescence (IF) staining, and release of infectious particles up to 30 days after infection without obvious cytolysis. By double-IF staining, cytokeratin-containing cells were shown to be susceptible to CVB4. The persistence of CVB4 was associated with a significantly increased rate of TEC proliferation (up to 70%) after 20 days of culture and a significantly increased chronic production of immunoreactive interleukin-6 (IL-6), leukemia inhibitory factor, and granulocyte-macrophage colony-stimulating factor in supernatant after 3 days of culture. The CVB4 replication and the release of cytokines were not restricted to the CVB4 E2 diabetogenic strain and did not depend on the genetic background of the host; however, TEC were more responsive to CVB4 E2 than CVB4 JBV as far as the production of cytokines.

Human antibodies isolated from plasma by affinity chromatography increase the coxsackievirus B4-induced synthesis of interferon-alpha by human peripheral blood mononuclear cells in vitro.

Coxsackievirus B4 (CVB4) can be found in circulating blood of patients; however, the interaction of CVB4 with peripheral blood mononuclear cells (PBMCs) is poorly understood. CVB4 induced low levels of IFN-alpha synthesis in PBMCs from healthy donors. In contrast, preincubation of infectious CVB4 with plasma from these donors containing anti-CVB4 antibodies strongly enhanced the synthesis of IFN-alpha. IgG obtained from plasma by chromatography formed immune complexes with CVB4 and increased significantly the CVB4-induced production of IFN-alpha by PBMCs. These antibodies did not have a neutralizing effect on CVB4 infection of Hep-2 cells. The role of CVB and adenovirus receptor (CAR), FcgammaRII and FcgammaRIII in the increased synthesis of IFN-alpha induced by CVB4 preincubated with IgG was shown by inhibition with specific antibodies. The major interferon-alpha-producing cells in response to CVB4-IgG complexes were CD14(+) cells and monocyte-enriched PBMCs. With the latter, detection of IFN-alpha by immunostaining was positive whereas in monocyte-depleted PBMCs it was not. This study shows that CVB4-induced synthesis of IFN-alpha by PBMCs can be enhanced by an antibody-dependent mechanism through interactions between the virus, non-neutralizing antivirus antibodies, FcgammaRII and III and CAR.

Risk factors for the development of diabetes mellitus in chronic hepatitis C virus genotype 4 infection.

Background and Aim:  A high occurrence of type 2 diabetes (T2D) in patients with chronic hepatitis C virus (HCV) infection has been reported in Kuwait and other countries. However, HCV genotype 4 has been underrepresented in all previous studies. Our aim was to investigate the viral and host risk factors associated with the development of T2D in patients with chronic hepatitis C genotype 4 infection in the absence of liver fibrosis and steatosis.

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

ABSTRACT Coxsackievirus B4 (CVB4)-induced production of alpha interferon (IFN-α) 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-α 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°C from CVB4E2 (VP4CVB4) and CVB3 (VP4CVB3) but not H antigen preincubated with plasma from healthy subjects or patients inhibited the plasma-dependent enhancement of CVB4E2- and CVB3-induced IFN-α synthesis. There was no cross-reaction between VP4CVB4 and VP4CVB3 in the inhibiting effect. IFN-α 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-VP4CVB4). 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-α 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-α synthesis by PBMC.

MxA protein in capillary blood of children with viral infections.

Capillary blood of febrile children was lysed by using a lysis buffer containing ascorbic acid. MxA quantitation was performed by an immunochemiluminescent assay. The MxA values were significantly higher in capillary blood of infants with viral infections due to adenovirus (n = 5), rotavirus (n = 15), or respiratory syncytial virus (n = 28), than in capillary whole blood from infants with bacterial infections (n = 6) and healthy control patients (n = 20). A strong correlation was found between the MxA values in capillary whole blood and peripheral whole blood (r′ = 0.86, P < 0.0001, n = 48). The MxA values found at these two sites were compared with the levels of IFN‐α obtained by a dissociation enhanced lanthanide fluoroimmunoassay. A correlation between these two values was found. The results show that the combination of collection of blood by finger prick and specific immunochemiluminescent assay for MxA protein measurement may be of value for the diagnosis of viral infections in children. J. Med. Virol. 59:547–551, 1999.

Novel NSP4 genotype in a camel G10P[15] rotavirus strain

In this study a Kuwaiti camel rotavirus strain, RVA/Camel-wt/KUW/s21/2010/G10P[15], is characterized by sequencing and phylogenetic analysis. The strain had multiple genes with high nucleotide sequence similarities to ovine and bovine strains (VP2, ≤ 96%; NSP2 and NSP5, ≤ 97%, NSP3, ≤ 94%), or, to porcine strains (VP1, ≤ 89%). Other genes had moderate sequence similarities (VP4, ≤ 87%; VP6, ≤ 81%; VP7, ≤ 82%) with reference strains from ruminants. The NSP4 gene shared limited sequence identity (≤ 71%) with other mammalian and avian rotavirus NSP4 types, and was designated a novel genotype, E15. This study demonstrates genetic diversity in the outer capsid and some backbone genes of an old-world camelid rotavirus strain and uncovers its common evolutionary roots with strains from other ruminants.

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.

Prevalence of human papillomavirus among women with normal cervical cytology in Kuwait.

This study was undertaken to determine the prevalence and type specific distribution of human papillomavirus (HPV) in women with normal cervical cytology in Kuwait. The study is the first of its type in Kuwait and one of few in the Middle East. The age specific distribution of HPV types was determined in 3,011 ThinPrep samples taken from women seeking routine gynaecological care. ThinPrep samples were screened for HPV DNA by real‐time PCR. The type specific distribution of the viruses was determined by PCR‐based sequencing. The results showed that HPV DNA was detected in 71 women (2.4%), and 21 different HPV genotypes were detected, comprising eight high‐risk (HR) (16, 31, 33, 53, 56, 58, 66, and 73), seven low‐risk (LR) (6, 11, 54, 61, 70, 81, and 90), four intermediate‐risk (IR) (67, 82, 83, and 84) and HPV 102 and HPV 106. LR HPV types were found in 71.8% of infected samples, HR types in 32.3%, and IR types in 7%. With regard to age, 40.8% of all HPVs were found in women 30–39 years of age, 29.6% in women 40–49 years of age, 19.7% in women over 50 years and 9.9% in women less than 34 years old. The study shows that the prevalence of HPV infection in Kuwait is among the lowest in the world and suggests that HPV vaccine could prevent the development of HPV associated cervical cancer in 1.39% of young females living in Kuwait. However, more extensive population‐based studies should be undertaken before implementing HPV vaccination. J. Med. Virol. 83:453–460, 2011.