A novel rat CVB1-VP1 monoclonal antibody 3A6 detects a broad range of enteroviruses

Saarinen, Niila V. V.; Laiho, Jutta E.; Richardson, Sarah J.; Zeissler, Marie; Stone, Virginia M.; Marjomäki, Varpu; Kantoluoto, Tino; Horwitz, Marc S.; Sioofy‐Khojine, Amir‐Babak; Honkimaa, Anni; Hankaniemi, Minna M.; Flodström‐Tullberg, Malin; Hyöty, Heikki; Hytönen, Vesa P.; Laitinen, Olli H.

doi:10.1038/s41598-017-18495-4

Cited by 21 publications

(23 citation statements)

References 35 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The 2A-1 and 3C antibodies recognized bands with the expected size of 2A pro and 3C pro , respectively, in cell lysates from all six CVB serotypes (Figure 1 D). The VP1 antibody, as expected, detected all six serotypes however, of note, the recognition of CVB6 was weaker than the other serotypes, an observation which we have also previously documented (Saarinen et al, 2018). Contrastingly, both protease antibodies demonstrated a robust detection of CVB6.…”

Section: Protease Antibodies Detect Cvb1-6 Infections By Western Blotsupporting

confidence: 85%

New Coxsackievirus 2Apro and 3Cpro protease antibodies for virus detection and discovery of pathogenic mechanisms

Laitinen

Svedin

Kapell

et al. 2018

Journal of Virological Methods

Self Cite

View full text Add to dashboard Cite

Enteroviruses (EVs), such as the Coxsackie B-viruses (CVBs), are common human pathogens, which can cause severe diseases including meningitis, myocarditis and neonatal sepsis. EVs encode two proteases (2A and 3C), which perform the proteolytic cleavage of the CVB polyprotein and also cleave host cell proteins to facilitate viral replication. The 2A cause direct damage to the infected heart and tools to investigate 2A and 3C expression may contribute new knowledge on virus-induced pathologies. Here, we developed new antibodies to CVB-encoded 2A and 3C; Two monoclonal 2A antibodies and one 3C antibody were produced. Using cells infected with selected viruses belonging to the EV A, B and C species and immunocytochemistry, we demonstrate that the 3C antibody detects all of the EV species B (EV-B) viruses tested and that the 2A antibody detects all EV-B viruses apart from Echovirus 9. We furthermore show that the new antibodies work in Western blotting, immunocyto- and immunohistochemistry, and flow cytometry to detect CVBs. Confocal microscopy demonstrated the expression kinetics of 2A and 3C, and revealed a preferential cytosolic localization of the proteases in CVB3 infected cells. In summary, the new antibodies detect proteases that belong to EV species B in cells and tissue using multiple applications.

show abstract

Section: Protease Antibodies Detect Cvb1-6 Infections By Western Blotsupporting

confidence: 85%

New Coxsackievirus 2Apro and 3Cpro protease antibodies for virus detection and discovery of pathogenic mechanisms

Laitinen

Svedin

Kapell

et al. 2018

Journal of Virological Methods

Self Cite

View full text Add to dashboard Cite

show abstract

“…The VP1 antigen (CVB3, strain Nancy, original sequence from GenBank: M33854.1) were prepared as described in [20]. The production of recombinant proteases 3C and 2A (CVB3, strain Nancy, original sequence from GenBank: M33854.1) was carried out as in [21].…”

Section: Preparation Of Elisa Antigensmentioning

confidence: 99%

“…The VP1 antigen (CVB3, strain Nancy) were prepared as described in [20]. Briefly, attL-flanked His-tagged VP1 protein coding synthetic genes were ordered from Life technologies and Gateway cloned into pBVboostFGII-vector [31,32] for antigen production.…”

Section: Appendix A2 Histologymentioning

confidence: 99%

Antibody Responses against Enterovirus Proteases are Potential Markers for an Acute Infection

Saarinen

Stone

Hankaniemi

et al. 2020

Viruses

Self Cite

View full text Add to dashboard Cite

Background: Enteroviruses are a group of common non-enveloped RNA viruses that cause symptoms ranging from mild respiratory infections to paralysis. Due to the abundance of enterovirus infections it is hard to distinguish between on-going and previous infections using immunological assays unless the IgM fraction is studied. Methods: In this study we show using Indirect ELISA and capture IgM ELISA that an IgG antibody response against the nonstructural enteroviral proteins 2A and 3C can be used to distinguish between IgM positive (n = 22) and IgM negative (n = 20) human patients with 83% accuracy and a diagnostic odds ratio of 30. Using a mouse model, we establish that the antibody response to the proteases is short-lived compared to the antibody response to the structural proteins in. As such, the protease antibody response serves as a potential marker for an acute infection. Conclusions: Antibody responses against enterovirus proteases are shorter-lived than against structural proteins and can differentiate between IgM positive and negative patients, and therefore they are a potential marker for acute infections.

show abstract

“…The 'unbiased proteomic analysis' claimed in the article by Dunne et al to verify the presence of viral proteins in type 1 diabetic pancreases sounds interesting but is based on unpublished data only, and it is not clear whether the presence of VP1 correlates with IHC positivity or type 1 diabetes. A number of novel antibodies with demonstrated high specificity for structural [15,17] and non-structural [18] enterovirus proteins have been developed but, to date, none of these have been used to confirm the presence of enterovirus protein in the pancreas of individuals with type 1 diabetes.…”

mentioning

confidence: 99%

Large enteroviral vaccination studies to prevent type 1 diabetes should be well founded and rely on scientific evidence

Skog

Klingel²,

Roivainen

et al. 2019

Diabetologia

View full text Add to dashboard Cite

Abbreviations IHC Immunohistochemistry PKR Protein kinase R VP1 Viral protein 1To the Editor: We read with great interest the For Debate article by Dunne et al [1] and fully agree with the conclusion that intervention studies using a vaccine or antiviral drugs represent the way forward to finally establish whether enterovirus plays a role in type 1 diabetes. While we welcome and support such initiatives, we consider it crucial that funders investing in the huge vaccination trials required to prove or disprove the link are provided with solid scientific evidence. Also, in the risk-benefit analysis and ethical considerations, it is essential that the available evidence is viewed critically. Therefore, we feel compelled to point out some issues regarding the evidence cited in the article by Dunne et al.First, the sequence data of the enterovirus isolated by Dotta et al from an individual with recent-onset type 1 diabetes [2] shared 99% identity with the coxsackievirus B4 prototype strain JVB Benschoten, originally isolated in 1951, clearly indicating that it was in fact a laboratory contaminant. This is well known and non-controversial among virologists and others within the field [3-5] and we find it problematic that this paper is uncritically cited as supporting evidence for the presence of pancreatic enterovirus in type 1 diabetes.Second, in the Diabetes Virus Detection (DiViD) study, we detected enterovirus RNA with RT-PCR in pancreatic tissue samples from only one of the six patients with recent-onset type 1 diabetes [6]. This patient was also RT-PCR-positive for enterovirus RNA in peripheral blood mononuclear cells, suggesting that the positive signal could have come from infected circulating blood cells present in the pancreas, particularly given that this biopsy was collected as a surgical specimen and not perfused with preservation solution to remove blood, as is usually done during organ procurement for transplantation. The reported, and frequently cited, enterovirus RNA-positive samples from four out of six DiViD patients were from the culture medium of isolated islets. These islets were isolated from the tip of the pancreatic tail under non-sterile conditions in a research laboratory for the purpose of functional islet studies [7]. The enterovirus-negative islets from six non-diabetic organ donors [6], referred to by Dunne et al, were isolated in our Good Manufacturing Process (GMP) facility for the purpose of clinical islet transplantation and, therefore, these results cannot be used to rule out the possibility of viral contamination in the culture medium of islets isolated from the type 1 diabetic individuals. Also, the discrepancy in PCR positivity between tissue samples and the culture medium of isolated islets is difficult to explain by a difference in sensitivity [8], and the possibility that the positive signal was derived from contaminating viral sequences introduced during islet isolation or culture cannot be excluded. Other studies (not cited in the article by Dunne et al) have failed to find e...

show abstract

A novel rat CVB1-VP1 monoclonal antibody 3A6 detects a broad range of enteroviruses

Cited by 21 publications

References 35 publications

New Coxsackievirus 2Apro and 3Cpro protease antibodies for virus detection and discovery of pathogenic mechanisms

New Coxsackievirus 2Apro and 3Cpro protease antibodies for virus detection and discovery of pathogenic mechanisms

Antibody Responses against Enterovirus Proteases are Potential Markers for an Acute Infection

Large enteroviral vaccination studies to prevent type 1 diabetes should be well founded and rely on scientific evidence

Contact Info

Product

Resources

About