Immunohistochemical detection of enteroviruses in pancreatic tissues of patients with type 1 diabetes using a polyclonal antibody against 2A protease of Coxsackievirus

Abstract

Aims/Introduction: The need for antiserum for immunohistochemical (IHC) detection of enterovirus (EV) in formaldehyde-fixed and paraffin-embedded samples is increasing. The gold standard monoclonal antibody (clone 5D8/1) against EV-envelope protein (VP1) was proven to cross-react with other proteins. Another candidate marker of EV proteins is 2A protease (2A pro ), which is encoded by the EV gene and translated by the host cells during EV replication, and participates processing proproteins to viral capsid proteins. Materials and Methods: We raised polyclonal antiserum by immunizing a rabbit with an 18-mer peptide of Coxsackievirus B1 (CVB1)-2A pro , and examined the specificity and sensitivity for EV on formaldehyde-fixed and paraffin-embedded tissue samples. Results: Enzyme-linked immunosorbent assay study showed a high titer of antibody for 18-mer peptide of CVB1-2A pro , cross-reacting with CVB3-2A pro peptide. IHC showed that antiserum against 2A pro reacted with CVB1-infected and VP1-positive Vero cells. Confocal laser scanning microscopy showed that antigen stained by the 2A pro antibody located in the same cell with VP1 stained by 5D8/1. IHC using 2A pro antiserum showed dense staining in the islets of EV-associated fulminant type 1 diabetes pancreas and that located in the same cell stained positive for VP1 (5D8/1). Specificity of 2A pro antiserum by IHC staining was confirmed by negative 2A pro in 14 VP1-negative non-diabetes control pancreases. Conclusions: Our study provides a new polyclonal antiserum against CVB1-2A pro , which might be useful for IHC of EV-infected human tissues stored as archive of formaldehyde-fixed and paraffin-embedded tissue samples.

Bi-glandular and persistent enterovirus infection and distinct changes of the pancreas in slowly progressive type 1 diabetes mellitus

“…As another positive control for VP1 antisera (5D8/1) by IHC, formalin-fixed and paraffin-embedded pancreas from FT1DM, which was ascertained by in situ hybridization 41 , was stained for VP1 (5D8/1) and was positive for enterovirus. New antisera against EV-2A pro (ET2112) were raised, and it was confirmed that the antiserum stained CVB1 in virus-infected Vero cells and also stained VP1-positive islet cells of FT1DM 40 , 41 . In this study, this antiserum was absorbed with 2A pro peptide of CVB1 at 10 ng/ml in SPIDDM (Supplemental Fig.…”

“…The 5D8/1 and 6E9-2 antibodies when incubated overnight with VP1 protein showed complete abolition of immunostaining for VP1 (Supplemental Fig. S 8a–h) 39 , 40 . Ten consecutive pancreatic sections in each case from 12 SPIDDM pancreases were stained for VP1 using 5D8/1 and 6E9-2.…”

“…As positive and negative controls for VP1 (5D8/1) by IHC, cultured Vero cells (green monkey kidney cells) that were infected or not infected by CVB1 (strain number: 16-20289), which was isolated from a Japanese child with aseptic meningitis in 2016, were used 40 . Vero cells at 0.1 and 3.0 multiplicity of infection (MOI)-infected and incubated for 24 h after infection and fixed by 5% formaldehyde were stained positive for VP1 40 . Mock cultured cells did not show positive staining for VP1 40 .…”

“…Vero cells at 0.1 and 3.0 multiplicity of infection (MOI)-infected and incubated for 24 h after infection and fixed by 5% formaldehyde were stained positive for VP1 40 . Mock cultured cells did not show positive staining for VP1 40 . As another positive control for VP1 antisera (5D8/1) by IHC, formalin-fixed and paraffin-embedded pancreas from FT1DM, which was ascertained by in situ hybridization 41 , was stained for VP1 (5D8/1) and was positive for enterovirus.…”

“…Expressions of 2A pro , MDA5, IFN-β1, and CXCL10 in islets were examined using antisera (Supplemental Table 1 ), as previously described 40 , 43 , 44 . All images were quantified using cellSens Dimension software (version 1.16; Olympus), and the values are expressed as the percentages of the positive islet areas for 2A pro , MDA5, IFN-β1, or CXCL10 divided by the respective islet or exocrine pancreatic area.…”