Genetic Adaptation of Coxsackievirus B1 during Persistent Infection in Pancreatic Cells

Coxsackie B (CVB) viruses have been associated with type 1 diabetes. We have recently observed that CVB1 was linked to the initiation of the autoimmune process leading to type 1 diabetes in Finnish children. Viral persistency in the pancreas is currently considered as one possible mechanism. In the current study persistent infection was established in pancreatic ductal and beta cell lines (PANC-1 and 1.1B4) using four different CVB1 strains, including the prototype strain and three clinical isolates. We sequenced 5′ untranslated region (UTR) and regions coding for structural and non-structural proteins and the second single open reading frame (ORF) protein of all persisting CVB1 strains using next generation sequencing to identify mutations that are common for all of these strains. One mutation, K257R in VP1, was found from all persisting CVB1 strains. The mutations were mainly accumulated in viral structural proteins, especially at BC, DE, EF loops and C-terminus of viral capsid protein 1 (VP1), the puff region of VP2, the knob region of VP3 and infection-enhancing epitope of VP4. This showed that the capsid region of the viruses sustains various changes during persistency some of which could be hallmark(s) of persistency

Detection of Viral −RNA and +RNA Strands in Enterovirus-Infected Cells and Tissues

The current methods to study the distribution and dynamics of viral RNA molecules inside infected cells are not ideal, as electron microscopy and immunohistochemistry can only detect mature virions, and quantitative real-time PCR does not reveal localized distribution of RNAs. We demonstrated here the branched DNA in situ hybridization (bDNA ISH) technology to study both the amount and location of the emerging −RNA and +RNA during acute and persistent enterovirus infections. According to our results, the replication of the viral RNA started 2–3 h after infection and the translation shortly after at 3–4 h post-infection. The replication hotspots with newly emerging −RNA were located quite centrally in the cell, while the +RNA production and most likely virion assembly took place in the periphery of the cell. We also discovered that the pace of replication of −RNA and +RNA strands was almost identical, and −RNA was absent during antiviral treatments. ViewRNA ISH with our custom probes also showed a good signal during acute and persistent enterovirus infections in cell and mouse models. Considering these results, along with the established bDNA FISH protocol modified by us, the effects of antiviral drugs and the emergence of enterovirus RNAs in general can be studied more effectively

Persistent coxsackievirus B1 infection triggers extensive changes in the transcriptome of human pancreatic ductal cells.

Enteroviruses, particularly the group B coxsackieviruses (CVBs), have been associated with the development of type 1 diabetes. Several CVB serotypes establish chronic infections in human cells in vivo and in vitro. However, the mechanisms leading to enterovirus persistency and, possibly, beta cell autoimmunity are not fully understood. We established a carrier-state-type persistent infection model in human pancreatic cell line PANC-1 using two distinct CVB1 strains and profiled the infection-induced changes in cellular transcriptome. In the current study, we observed clear changes in the gene expression of factors associated with the pancreatic microenvironment, the secretory pathway, and lysosomal biogenesis during persistent CVB1 infections. Moreover, we found that the antiviral response pathways were activated differently by the two CVB1 strains. Overall, our study reveals extensive transcriptional responses in persistently CVB1-infected pancreatic cells with strong opposite but also common changes between the two strains. </p

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

This is the author accepted manuscript. The final version is available from Springer Nature via the DOI in this record.Enteroviruses (EVs) are common RNA viruses that cause diseases ranging from rash to paralytic poliomyelitis. For example, EV-A and EV-C viruses cause hand-foot and mouth disease and EV-B viruses cause encephalitis and myocarditis, which can result in severe morbidity and mortality. While new vaccines and treatments for EVs are under development, methods for studying and diagnosing EV infections are still limited and therefore new diagnostic tools are required. Our aim was to produce and characterize new antibodies that work in multiple applications and detect EVs in tissues and in vitro. Rats were immunized with Coxsackievirus B1 capsid protein VP1 and hybridomas were produced. Hybridoma clones were selected based on their reactivity in different immunoassays. The most promising clone, 3A6, was characterized and it performed well in multiple techniques including ELISA, immunoelectron microscopy, immunocyto- and histochemistry and in Western blotting, detecting EVs in infected cells and tissues. It recognized several EV-Bs and also the EV-C representative Poliovirus 3, making it a broad-spectrum EV specific antibody. The 3A6 rat monoclonal antibody can help to overcome some of the challenges faced with commonly used EV antibodies: it enables simultaneous use of mouse-derived antibodies in double staining and it is useful in murine models.This study was supported by TEKES – the Finnish Funding Agency for Innovation (project THERDIAB 1843/31/2014) as well as JDRF grants for the nPOD-Virus Group, JDRF 25-2012-516 to A. Pugliese and JDRF 25-2012-770 to M.A. Atkinson for the nPOD-Virus Group, the Diabetes Research Foundation in Finland, the Sigrid Juselius Foundation, Reino Lahtikari Foundation, the Academy of Finland and the European Commission (Persistent Virus Infection in Diabetes Network (PEVNET), Frame Programme 7, Contract No. 261441) and the Swedish Child Diabetes Research Foundation. Additional support was given by a Diabetes Research Wellness Foundation Non-Clinical Research Fellowship and, since 2014, a JDRF Career Development Award (5-CDA-2014-221-A-N) to S.J.R

Vemurafenib Inhibits Acute and Chronic Enterovirus Infection by Affecting Cellular Kinase Phosphatidylinositol 4-Kinase Type IIIb

Enteroviruses are one of the most abundant viruses causing mild to serious acute infections in humans and also contributing to chronic diseases like type 1 diabetes. Presently, there are no approved antiviral drugs against enteroviruses. Here, we studied the potency of vemurafenib, an FDA-Approved RAF kinase inhibitor for treating BRAFV600E mutant-related melanoma, as an antiviral against enteroviruses. We showed that vemurafenib prevented enterovirus translation and replication at low micromolar dosage in an RAF/MEK/ERK-independent manner. Vemurafenib was effective against group A, B, and C enteroviruses, as well as rhinovirus, but not parechovirus or more remote viruses such as Semliki Forest virus, adenovirus, and respiratory syncytial virus. The inhibitory effect was related to a cellular phosphatidylinositol 4-kinase type IIIb (PI4KB), which has been shown to be important in the formation of enteroviral replication organelles. Vemurafenib prevented infection efficiently in acute cell models, eradicated infection in a chronic cell model, and lowered virus amounts in pancreas and heart in an acute mouse model. Altogether, instead of acting through the RAF/MEK/ERK pathway, vemurafenib affects the cellular PI4KB and, hence, enterovirus replication, opening new possibilities to evaluate further the potential of vemurafenib as a repurposed drug in clinical care. IMPORTANCE Despite the prevalence and medical threat of enteroviruses, presently, there are no antivirals against them. Here, we show that vemurafenib, an FDA-Approved RAF kinase inhibitor for treating BRAFV600E mutant-related melanoma, prevents enterovirus translation and replication. Vemurafenib shows efficacy against group A, B, and C enteroviruses, as well as rhinovirus, but not parechovirus or more remote viruses such as Semliki Forest virus, adenovirus, and respiratory syncytial virus. The inhibitory effect acts through cellular phosphatidylinositol 4-kinase type IIIb (PI4KB), which has been shown to be important in the formation of enteroviral replication organelles. Vemurafenib prevents infection efficiently in acute cell models, eradicates infection in a chronic cell model, and lowers virus amounts in pancreas and heart in an acute mouse model. Our findings open new possibilities to develop drugs against enteroviruses and give hope for repurposing vemurafenib as an antiviral drug against enteroviruses

Antiviral Properties of Chemical Inhibitors of Cellular Anti-Apoptotic Bcl-2 Proteins

Viral diseases remain serious threats to public health because of the shortage of effective means of control. To combat the surge of viral diseases, new treatments are urgently needed. Here we show that small-molecules, which inhibit cellular anti-apoptotic Bcl-2 proteins (Bcl-2i), induced the premature death of cells infected with different RNA or DNA viruses, whereas, at the same concentrations, no toxicity was observed in mock-infected cells. Moreover, these compounds limited viral replication and spread. Surprisingly, Bcl-2i also induced the premature apoptosis of cells transfected with viral RNA or plasmid DNA but not of mock-transfected cells. These results suggest that Bcl-2i sensitizes cells containing foreign RNA or DNA to apoptosis. A comparison of the toxicity, antiviral activity, and side effects of six Bcl-2i allowed us to select A-1155463 as an antiviral lead candidate. Thus, our results pave the way for the further development of Bcl-2i for the prevention and treatment of viral diseases.Peer reviewe

Antiviral properties of chemical inhibitors of cellular anti-apoptotic Bcl-2 proteins

Viral diseases remain serious threats to public health because of the shortage of effective means of control. To combat the surge of viral diseases, new treatments are urgently needed. Here we show that small-molecules, which inhibit cellular anti-apoptotic Bcl-2 proteins (Bcl-2i), induced the premature death of cells infected with different RNA or DNA viruses, whereas, at the same concentrations, no toxicity was observed in mock-infected cells. Moreover, these compounds limited viral replication and spread. Surprisingly, Bcl-2i also induced the premature apoptosis of cells transfected with viral RNA or plasmid DNA but not of mock-transfected cells. These results suggest that Bcl-2i sensitizes cells containing foreign RNA or DNA to apoptosis. A comparison of the toxicity, antiviral activity, and side effects of six Bcl-2i allowed us to select A-1155463 as an antiviral lead candidate. Thus, our results pave the way for the further development of Bcl-2i for the prevention and treatment of viral diseases.</p

Genetic Adaptation of Coxsackievirus B1 during Persistent Infection in Pancreatic Cells

Coxsackie B (CVB) viruses have been associated with type 1 diabetes. We have recently observed that CVB1 was linked to the initiation of the autoimmune process leading to type 1 diabetes in Finnish children. Viral persistency in the pancreas is currently considered as one possible mechanism. In the current study persistent infection was established in pancreatic ductal and beta cell lines (PANC-1 and 1.1B4) using four different CVB1 strains, including the prototype strain and three clinical isolates. We sequenced 5&prime; untranslated region (UTR) and regions coding for structural and non-structural proteins and the second single open reading frame (ORF) protein of all persisting CVB1 strains using next generation sequencing to identify mutations that are common for all of these strains. One mutation, K257R in VP1, was found from all persisting CVB1 strains. The mutations were mainly accumulated in viral structural proteins, especially at BC, DE, EF loops and C-terminus of viral capsid protein 1 (VP1), the puff region of VP2, the knob region of VP3 and infection-enhancing epitope of VP4. This showed that the capsid region of the viruses sustains various changes during persistency some of which could be hallmark(s) of persistency

Molecular Markers and Drug Treatment for Persistent Enterovirus Infection in Pancreatic Cell Lines

Enterovirukset aiheuttavat laajan kirjon erilaisia sairauksia, vaihdellen lievästä nuhakuumeesta vakavampiin sairauksiin, kuten aivo-, sydänlihas- ja aivokalvontulehdukseen. Lisäksi enterovirukset ovat liitetty kroonisiin sairauksiin, kuten polion jälkeiseen oireyhtymään, keliakiaan, krooniseen sydänlihaksen rappeumaan ja tyypin 1 diabetekseen. Tyypin 1 diabeteksessa insuliinia tuottavat haiman β-solut tuhoutuvat, mikä johtaa insuliinituotannon häiriöön ja verensokerin säätelyn häiriöön. Sairautta hoidetaan päivittäin annettavilla insuliinipistoksilla ja sitä ei pystytä ennaltaehkäisemään. Nykykäsityksen mukaan geneettiset tekijät määräävät yksilön alttiuden sairastua tyypin 1 diabetekseen, mutta monet eri ympäristötekijät voivat määrätä sen kenelle tauti puhkeaa. Yhteys enterovirusten ja tyypin 1 diabeteksen välillä on havaittu lukuisissa epidemiologisissa tutkimuksissa. Enteroviruksia tunnetaan yli 100 erilaista, ja tämä yhteys on liitetty erityisesti yhteen virusten alaryhmään, Coxsackie B - viruksiin (CVB). Näyttää siltä, että nämä virukset hakeutuvat haiman β-soluihin ja voivat aiheuttavat siellä pitkittyneen (persistoivan) infektion, joka johtaa solujen tuhoutumiseen joko suoraan viruksen vaikutuksesta tai immuunivasteen välityksellä. Kuitenkin syy-yhteys enterovirusten ja tyypin 1 diabeteksen välillä on vielä vailla lopullista varmistusta. Tämän tutkimuksen tavoitteena oli kehittää persistoivan Coxsackievirus B1 (CVB1) infektion solumalli haimasolulinjoissa. Viruksen läsnäolo osoitettiin toteamalla viruksen genomia ja virusproteiineja soluviljelmästä. Persistoivan infektion synnyn kannalta oleellisia mekanismeja tarkasteltiin tutkimalla sekä isäntäsolussa että viruksessa tapahtuvia muutoksia. Tutkimuksessa testattiin myös viruslääkkeiden tehoa akuuttiin ja persistoivaan CVB infektioon solumalleissa. Persistoivan infektion aikana tapahtuvia solutoiminnan muutoksia kartoitettiin proteomiikan menetelmillä ja viruksessa tapahtuvia muutoksia sekvensoimalla koko viruksen koko genomi uuden sukupolven sekvensointimenetelmällä (NGS) infektion eri vaiheissa. Viruslääkkeiden tehoa tutkittiin sekä akuutin CVB infektion solumallissa käyttäen kaikkia kuutta CVB serotyyppiä että persistoivan CVB infektion käyttäen kahta eri CVB1 kantaa ja kahta haimasolulinjaa. Persistoivan CVB1 infektion solumalli luokiteltiin kantajatyyppiseksi persistenssiksi, sillä virusta oli viljelmässä suuria määriä, vaikka vain osa soluista oli infektoituneita. Solun sisäisten proteiinien ilmentymisessä ja niiden erityksessä tapahtui merkittäviä muutoksia persistoivan CVB1 infektion aikana. CVB1 viruksen käyttämän Coxsackie- ja adenovirusreseptorin (CAR) sekä mitokondrioiden energia- aineenvaihduntaan ja solujen eritystoiminnan säätelyyn liittyvien proteiinien ilmentyminen laski selvästi persistoivan infektion aikana. Persistoiva CVB1-infektio vaikutti myös β-solujen toimintaa sääteleviin proteiineihin, sekä tyypin 1 diabetekseen yhdistettyihin proteiineihin. Solun proteiinien ilmentymisessä havaittiin selviä eroja CVB1 viruskantojen välillä. Esimerkiksi solun luontaisen immuniteetin aktivaatiossa oli suuria eroja: ATCC kannan aiheuttama persistoiva CVB1 infektio stimuloi näitä antiviraalivasteita kun taas 10796 kannan aiheuttama infektio hiljensi niitä. Persistoivan CVB1 infektion aikana kehittyneet mutaatiot, jotka johtivat aminohapon muutokseen, sijoittuivat pääosin viruksen rakenneproteiineja koodaaville alueille. Tutkimuksessa sekvensoitiin kahdeksan virusta, jotka olivat peräisin CVB1 kantojen aiheuttamista persistoivista infektioista. Yksi mutaatio, K257R, viruksen VP1-proteiinissa, löydettiin kaikista kahdeksasta sekvensoidusta viruksesta. Persistoiva CVB1-infektio pystyttiin häätämään soluista joillakin tutkituista viruslääkkeistä. Fluoksetiini, masennuslääke, jolla on havaittu vaikutusta Enteroviruksiin, ja Hizentra, immunoglobuliinituote, joka sisältää neutraloivia vasta- aineita CVB1 viruksia kohtaan, pystyivät häätämään sekä ATCC että 10796 kannan aiheuttaman persistoivan CVB1 infektion haimasoluista. Pleconaril ja Enviroksiimi pystyivät myös häätämään persistoivan ATCC kannan aiheuttaman infektion. Yhteenvetona voidaan todeta, että kehitetyt persistoivan CVB1 infektion haimasolumallit edustavat kantajatyyppistä enteroviruksen persistenssiä. Persistoiva CVB1 infektio johti merkittäviin muutoksiin solun proteiinin ilmentymisessä ja erityksessä Ei-synonyymiset mutaatiot esiintyvät viruksen rakenneproteiineiss a useammin kuin ei-rakenteellisissa proteiineissa. Erityisesti yksi mutaatio VP1- proteiinissa, K257R, havaittiin kaikissa persistoivan infektion aikana muodostuneissa viruksissa. Persistoiva infektio pystyttiin häätämään Fluoksetiinilla ja Hizentra immunoglobuliinituotteella. Tutkimukset tulokset tuovat uutta tietoa persistoivan enterovirusinfektion mekanismeista ja viruslääkkeistä, joiden avulla tämäntyyppinen infektio voidaan parantaa solumalleissa. Tulosten pohjalta voidaan tulevaisuudessa pyrkiä etsimään keinoja persistoivan enterovirusinfektion ja siihen liittyvien sairauksien ehkäisyyn ja hoitoon.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