Enteroviruses (EVs) cause a wide range of different diseases from the common cold to more severe illnesses such as encephalitis, myocarditis, and meningitis. Additionally, EVs have been associated with chronic diseases, including post-polio syndrome, coeliac disease, dilated cardiomyopathy, and type 1 diabetes (T1D). In T1D, insulin producing pancreatic β-cells are destroyed, leading to a disorder of insulin production and secretion, thereby a failure to regulate blood sugar. T1D is treated by daily insulin injections, and no preventable means exist.
Genetical factors determine the individual’s susceptibility to develop T1D, but environmental factors play an important role in the pathogenesis. EVs are associated with the development of T1D in numerous epidemiological studies. More than 100 different EVs are known, and the association with diabetes is especially linked with one subgroup of EVs, Coxsackie B viruses (CVBs). It seems that these viruses target pancreatic β-cells and can cause persistent infection that gradually leads to cell destruction either by the virus or through an immune response. However, the causal relationship between enteroviruses and type 1 diabetes is yet to be conclusively confirmed.
The aim of this study was to establish Coxsackievirus B1 (CVB1) persistent infection in pancreatic cell lines. The presence of the virus in these cells was verified by detecting viral RNA and proteins from infected cells. The mechanisms of viral persistency were further investigated by studying cellular and viral changes during the development of persistent infections. The entire proteome of persistently infected cells was compared to non-infected cells and the entire genome of the virus was sequenced using a next-generation sequencing (NGS) technology. In addition, ten antiviral drugs were tested for their efficacy against CVB viruses in acute infection models and for their ability to cure persistent CVB infection from pancreatic cell lines.
Established persistent CVB1 infections showed features that are characteristic of a carrier state type of persistency, including the production of virus progenies in high titers and infection in only a small number of cells. The expression of several intracellular and secreted proteins changed dramatically during persistent CVB1 infection compared to uninfected cells.
The Coxsackie and adenovirus receptor (CAR), which is used by CVB1 for internalization was strongly downregulated. Also, the proteins that are associated with mitochondrial energy metabolism and regulated secretion pathways were downregulated. Persistent CVB1 infection also influenced proteins that are associated with β-cell fate and T1D associated proteins. Additionally, the changes in cellular proteome differed between the two CVB1 strains that were used to establish persistent infection. Most strikingly, the antiviral innate immune response proteins were strongly upregulated in cells persistently infected by the ATCC prototype CVB1 strain but downregulated in cells infected by the wild-type 10796 CVB1 strain.
The NGS analysis of eight persistent infection-derived viruses (PIDVs) representing different CVB1 parental strains identified multiple mutations that led to amino acid substitutions in PIDVs. The majority of these mutations were located in structural virus proteins. One mutation was exclusively associated with the development of persistency (K257R in VP1 protein) as it was discovered in all PIDVs.
Certain antiviral drugs were able to eradicate persistent CVB1 infections from pancreatic cell lines. Fluoxetine, an antidepressant drug, which has been shown to have some anti-EV activity, and Hizentra, which is an immunoglobulin concentrate, with neutralizing antibodies against CVB1 were able to eradicate persistent infection of both CVB1 strains (ATCC and 10796). Pleconaril and Enviroxime were only able to eradicate the CVB1 ATCC strain.
In conclusion, the persistent CVB1 infection models that were established in this study represented the carrier state type of persistency. Persistent CVB1 infection markedly changed the cellular protein expression and secretion in pancreatic cells. Non-synonymous mutations appeared in the viral structural proteins more often than in the non-structural proteins during the development of CVB1 persistency. One specific mutation in the VP1 protein, K257R, occurred in all PIDVs. This type of persistent infection could also be eradicated by certain antiviral drugs, particularly Fluoxetine and immunoglobulin concentrate.
Altogether, the study generated new information about the virus-cell interactions that play a role in the development of persistent CVB infections and antiviral drugs potentially effective in their treatment.
Based on the results, future efforts can be made to find ways to prevent and treat persistent enterovirus infections and related diseases.
The doctoral dissertation of M.Sc. Anni Honkimaa in the field of virology titled Molecular markers and drug treatment for persistent enterovirus infection in pancreatic cell lines will be publicly examined at the Faculty of Medicine and Health Technology of Tampere University at 12 o'clock on Friday 1 October, 2021. The venue is Arvo building auditorium F115, address: Arvo Ylpön katu 34. Docent Carita Savolainen-Kopra from Finnish institute of health and welfare will be the opponent while Professor Heikki Hyöty will act as the custos.
The event can be followed via remote connection from this link.
The dissertation is available online at
http://urn.fi/URN:ISBN:978-952-03-2103-1
Photo: Antti Takkinen