To determine the usefulness of nasal swabs as a simple method for detection of respiratory viruses, we compared nasal swabs and nasopharyngeal aspirates obtained at the same time from the opposite nostrils of 230 children with upper respiratory infection. The sensitivity of nasal swabs was comparable to that of nasopharyngeal aspirates for the detection of all major respiratory viruses except respiratory syncytial virus

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The effect of enterovirus infection on the appearance of T1D-associated autoimmunity seems to be modified by exposure to cow's milk in early infancy suggesting an interaction between these factors. Moreover, these results provide an explanation for the controversial findings obtained when analysing the effect of any single one of these factors on the appearance of T1D-associated autoimmunity.

SummaryRotavirus infections have been implicated as a possible trigger of type 1 diabetes. We elucidated this connection by comparing peripheral blood T cell responses to rotavirus between children with newly diagnosed type 1 diabetes ( n = = = = 43), healthy children with multiple diabetes-associated autoantibodies ( n = = = = 36) and control children carrying human leukocyte antigen (HLA)-conferred susceptibility to type 1 diabetes but without autoantibodies ( n = = = = 104). Lymphocyte proliferation assays based on stimulation with an antigen were performed using freshly isolated peripheral blood mononuclear cells (PBMC) and IgG and IgA class rotavirus antibodies were measured using plasma samples collected from the children. The expression of interferon (IFN)-γ γ γ γ , interleukin (IL)-4, IL-10 and transforming growth factor (TGF)-β β β β in PBMC was studied with real-time polymerase chain reaction (PCR) in a subgroup of 38 children. No differences were observed in the strength or frequency of positive T cell responses to rotavirus between children with overt diabetes, children with multiple autoantibodies and control children. Children with diabetesassociated autoantibodies had, instead, stronger T cell responses to purified coxsackie B4 virus than control children. Rotavirus-stimulated lymphocytes from autoantibody-positive children produced more IL-4 and phytohaemagglutinin (PHA)-stimulated lymphocytes more IL-4 and IFN-γ γ γ γ than lymphocytes from control children. PHA-stimulated lymphocytes from children with diabetes also produced more IL-4 and purified protein derivative (PPD)-stimulated lymphocytes less TGF-β β β β than lymphocytes from autoantibody-negative control children. In conclusion, our lymphocyte proliferation studies did not provide evidence supporting an association between rotavirus infections and the development of type 1 diabetes or diabetes-associated autoantibodies in young children.

Coxsackie B viruses (CBV) have been indicated as environmental triggers initiating autoimmune destruction of insulin-producing pancreatic beta-cells, and molecular mimicry might be the mechanism. A prime candidate for inducing cross-reactive immune responses is a homology sequence, PEVKEK, found both in CBV4 2C protein and in GAD65. To characterize the CBV4-specific T-cell epitopes, overlapping peptides covering the 2C protein were synthesized and CBV4-specific T-cell lines were established from healthy and diabetic subjects. The T-cell epitopes were dependent on the HLA-DR genotype of the T-cell donor, but no difference between diabetic and healthy subjects could be detected. Peptide p4, which included the PEVKEK sequence, contained an HLA-DR1-restricted T-cell epitope. Three randomly selected CBV4-specific T-cell lines, which responded to peptide p4, failed to recognize GAD65 protein or GAD65 peptides containing the PEVKEK sequence. We conclude that the CBV4 2C protein is strongly immunogenic for T-cells and PEVKEK is included in a T-cell epitope. However, presentation of this epitope in the context of neutral HLA-DR1 allele does not support its role in pathogenesis of type 1 diabetes.

To study humoral and cellular immunity against human parechovirus type 1 (HPEV1), the viral capsid proteins VP0, VP1, and VP3 were expressed and purified as glutathione S-transferase (GST)-tagged recombinant proteins. The fusion proteins were used to raise antisera in rabbits. VP0 and VP1 antisera specifically detected HPEV1-infected cells in culture by immunoperoxidase staining and immunofluorescence. Furthermore, antisera against the VP0 and VP1 proteins had neutralizing effects against HPEV1 infection. When the HPEV1 antibody titers of 20 adults and 55 children were determined by a microneutralization test, the prevalence of HPEV1 antibodies in the adult population was 96%, while 50% of children were seropositive. Selected sera were used to evaluate HPEV1 fusion proteins as antigens in an enzyme immunoassay. The VP3 capsid protein appeared to be suitable for the purpose, with specificity of 100% and sensitivity of 96% compared to the neutralization test. Furthermore, T-cell responses to the purified HPEV1 and HPEV1 capsid fusion proteins were studied in 20 adults. Sixty percent of the subjects had T-cell proliferation responses to purified HPEV1, and 90% of the subjects also had positive T-cell responses to at least one of the GST capsid proteins.Human parechovirus type 1 (HPEV1), the type member of the genus Parechovirus, is a common pathogen with a worldwide distribution (5, 9). Infections occur early in life, and seropositivity increases rapidly after 1 year of age. The most common clinical manifestations are gastroenteritis and respiratory infections, while central nervous system symptoms are more rare than in enterovirus infections. Parechoviruses form one of the nine genera in the family Picornaviridae and include two human pathogens, HPEV1 and HPEV2, and a rodent virus, Ljungan virus (12). Like other picornaviruses, parechoviruses are small nonenveloped particles containing an RNA genome with positive polarity and a protein capsid consisting of 60 copies of each of the capsid proteins VP1 to VP4 (8). However, parechoviruses have distinctive molecular properties compared to other picornaviruses, including differences in capsid protein processing (VP0 is not cleaved to VP2 and VP4) and functions of the nonstructural proteins (3,7,21).B-cell antigenic epitopes of some picornaviruses, like polioviruses and rhinoviruses, are relatively well known (22). The major antigenic sites in polioviruses exist in VP1 and, to a lesser extent, in VP2 and VP3 (15). There is a linear epitope in VP1, while amino acids participating in two conformational epitopes are scattered along the capsid proteins. It has been shown by using rabbit antibodies and synthetic peptides that in HPEV1 one immunogenic region resides in VP0 and another resides at the C terminus of VP1 (10). The latter region contains an RGD motif which participates in recognition of ␣V␤3 integrin on the host cell surface (21).Cellular immunity and T-cell epitopes of enteroviruses, like coxsackie B viruses (CBV) and polioviruses, have been studied by proliferat...

The present study aimed to characterize systematically the target epitopes of T cell responses in CBV4 structural proteins. These were studied by synthesizing 86 overlapping 20-aa-long peptides covering the known sequence of CBV4 structural proteins and analyzing the proliferation responses of 18 CBV4-specific T cell lines against these peptides. Recognized peptides differed depending on the HLA-DR genotype of the T cell donor. They were concentrated to the VP4 and VP2 regions as six of seven common peptide epitopes located in this region, whereas there was only one in the VP3 region and none in the VP1 region. Peptides from conserved areas were recognized more often (on average, 15% of them stimulated each T cell line) than those derived from variable areas (3%) (P < 0.0001, Fisher's exact test). Some conserved peptides inducing T cell responsiveness in most subjects were identified, a knowledge which can be useful in the development of new synthetic vaccines.