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Enteroviruses are one of the most abundant groups of viruses infecting humans, and yet there are no approved antivirals against them. To find effective antiviral compounds against enterovirus B group viruses, an in-house chemical library was screened. The most effective compounds against Coxsackieviruses B3 (CVB3) and A9 (CVA9) were CL212 and CL213, two N-phenyl benzamides. Both compounds were more effective against CVA9 and CL213 gave a better EC50 value of 1 µM with high a specificity index of 140. Both drugs were most effective when incubated directly with viruses suggesting that they mainly bound to the virions. A real-time uncoating assay showed that the compounds stabilized the virions and radioactive sucrose gradient as well as TEM confirmed that the viruses stayed intact. A docking assay, taking into account larger areas around the 2-and 3-fold axes of CVA9 and CVB3, suggested that the hydrophobic pocket gives the strongest binding to CVA9 but revealed another binding site around the 3-fold axis which could contribute to the binding of the compounds. Together, our data support a direct antiviral mechanism against the virus capsid and suggest that the compounds bind to the hydrophobic pocket and 3-fold axis area resulting in the stabilization of the virion.

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 IIIβ (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. peerReviewed

Tämä opinnäytetyö käsittelee tutkimustietoa tyypin 1 diabeteksen (T1D) patogeneesin hypoteesista, jonka mukaan kliiniseen sairauteen johtavan autoimmuunireaktion laukaisee haiman saarekkeiden virusinfektio. T1D on autoimmuunitauti, jossa haiman saarekkeiden beetasolut tuhoutuvat, jolloin niiden insuliinin tuotanto menetetään. Insuliinin tuotannon romahtaessa kehon energia-aineenvaihdunta häiriintyy vakavasti. Hoitamattomana tila johtaa kuolemaan. Menetetty haiman insuliinituotanto voidaan korvata insuliinivalmisteilla, mutta tästä huolimatta potilaat kärsivät merkittävästä elämänlaadun laskusta ja lyhyemmästä elinajanennusteesta. Lisäksi insuliinihoito on kallista. Haiman beetasoluja tuhoavan autoimmuunireaktion laukaisevat tekijät ja solutuhon mekanismit tunnetaan edelleenkin vain osittain. Tiedetään, että tietyt HLA-genotyypit altistavat T1D:n puhkeamiselle. Autoimmuuniprosessi alkaa jo kauan ennen kuin T1D voidaan kliinisesti todeta. Tätä ennen haiman saarekkeisiin hakeutuu lymfosyyttejä, jolloin kehittyy saarekkeiden tulehdusreaktio – insuliitti. Myös humoraalisen immuunijärjestelmän aktivoituminen beetasoluja vastaan voidaan usein havaita ennen T1D:n kliinistä ilmentymistä: Verenkierrossa todetaan autovasta-aineita beetasolujen proteiineja kohtaan. Autovasta-aineista tärkeimpiä ovat insuliini-, GAD65-, IA-2- ja ZnT8-vasta-aineet. Laajoissa epidemiologisissa tutkimuksissa on havaittu enterovirusten sukuun kuuluvien coxsackie B -virusten (CVB) olevan yhteydessä T1D:n puhkeamiseen. CVB:t tartuttavat pääasiassa hengitystie-eritteiden tai ulostekontaminaation välityksellä, ja niiden ensisijainen replikaatiopaikka on suolisto. Ne aiheuttavat etenkin lapsissa yleensä vähäoireisia tauteja, mutta ne voivat aiheuttaa myös hankalia monielininfektioita. CVB:ten on osoitettu infektoivan haiman saarekkeiden beetasoluja in vitro ja in vivo sekä kokeellisissa hiirimalleissa että ihmisissä. CVB pääsee beetasoluihin niiden solukalvollaan ilmentämän coxsackie-adenovirusreseptorin (CAR) välityksellä. Hiiritutkimuksissa on havaittu, että vain tietyt CVB:ten alalajit kykenevät aiheuttamaan beetasolujen infektion ja insuliininpuutosdiabeteksen kaltaisen taudin hiirillä. CVB4 E2 ja CVB4 JVB -viruskannat aiheuttavat insuliininpuutosdiabeteksen hiirille. Tutkimuksissa, joissa on käytetty muita CVB-kantoja, ei ole kyetty aiheuttamaan hiirten beetasolujen infektiota. Persistoivassa infektiossa virus ei infektion akuutin lyyttisen vaiheen jälkeen poistu kudoksesta. Todisteita tämänlaisesta matala-asteisesta enterovirusinfektiosta on havaittu vastikään T1D:een sairastuneiden haiman saarekkeista. Haiman saarekkeiden persistoivan CVB-infektion ajatellaan olevan yksi T1D:een johtavan autoimmuuniprosessin laukaiseva tekijä. Persistoivia CVB-infektioita on pystytty kokeellisesti aikaansaamaan ihmisen haimasolulinjoissa in vitro ja hiirillä myös in vivo. Vaikka selkeää kausaliteettia CVB-infektion ja T1D:n kehittymisen välillä ei ole vielä voitu osoittaa, nämä infektiot ovat tärkeä tutkimuskohde T1D:n patogeneesin ymmärtämisen kannalta. Virusten ja T1D:n yhteyden paremmin ymmärtäminen saattaa tulevaisuudessa mahdollistaa T1D:n ehkäisyn esimerkiksi rokotusten avulla. This thesis aims to highlight current evidence on one of the leading hypothesis of type 1 diabetes (T1D) pathogenesis, where the disease is caused by an autoimmune reaction triggered by viral infection in the pancreatic islets. T1D is an autoimmune disease, in which the beta cells within the pancreatic islets are destroyed and their insulin production is lost. The loss of insulin production leads to a crisis in body energy metabolism that, if left untreated, will lead to death. Even if treated with exogenous insulin therapy, T1D causes major decline in quality of life and life expectancy. Insulin treatment is also expensive. The autoimmune progress and the factors that lead to it are now well understood at the moment. It is known that certain HLA-genotypes predispose to T1D. The autoimmune process leading to T1D begins long before the condition manifests clinically. Before that, the pancreatic islets are infiltrated by lymphocytes, leading to an inflammation of the pancreatic islets – insulitis. Activation of the humoral immune system can also be detected before clinical T1D manifestation. Autoantibodies against beta cell proteins are detected in blood, most important being autoantibodies against insulin, GAD65, IA-2, and ZnT8. Coxsackie B viruses (CVBs) that belong in the genus Enterovirus have associated with T1D onset in large epidemiological studies. CVBs are transmitted mainly via respiratory or faecal-oral route and their primary replication site is the gastrointestinal tract. The symptoms of an infection are usually mild in children, but CVBs can also cause severe septic multiorgan infections. CVBs have been shown to infect pancreatic beta cells in vitro and in vivo in mouse models as well as in humans. CVBs can enter the beta cells via the Coxsackievirus and adenovirus receptor (CAR) on the cell surface. Mouse studies have shown that only select few CVB strains can infect beta cells and cause T1D-like syndrome in mice. CVB4 E2 and CVB4 JVB virus strains can cause T1D-like syndrome in mice. Other studies that have used other CVB strains have not been able to infect mouse beta cells. During persistent infection the virus is not cleared after the acute lytic phase. Evidence of persistent enterovirus infection have been found in the pancreases of recent-onset T1D patients. Persistent CVB infection of pancreatic islets is thought to be the factor triggering the autoimmune process leading to T1D. Persistent CVB infections have been established in human pancreatic cell models in vitro and in mouse models also in vivo. Although no definitive causality between CVB infections and the development of T1D has been proven as of to date, these infections are important focus point in the research of T1D pathogenesis. Better understanding of the association between T1D and viruses may help to prevent T1D using e.g., vaccines.

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.

Coxsackievirus B (CVB) enteroviruses are common pathogens that can cause acute and chronic myocarditis, dilated cardiomyopathy, aseptic meningitis, and they are hypothesized to be a causal factor in type 1 diabetes. The licensed enterovirus vaccines and those currently in clinical development are traditional inactivated or live attenuated vaccines. Even though these vaccines work well in the prevention of enterovirus diseases, new vaccine technologies, like virus-like particles (VLPs), can offer important advantages in the manufacturing and epitope engineering. We have previously produced VLPs for CVB3 and CVB1 in insect cells. Here, we describe the production of CVB3-VLPs with enhanced production yield and purity using an improved purification method consisting of tangential flow filtration and ion exchange chromatography, which is compatible with industrial scale production. We also resolved the CVB3-VLP structure by Cryo-Electron Microscopy imaging and single particle reconstruction. The VLP diameter is 30.9 nm on average, and it is similar to Coxsackievirus A VLPs and the expanded enterovirus cell-entry intermediate (the 135s particle), which is ~2 nm larger than the mature virion. High neutralizing and total IgG antibody levels, the latter being a predominantly Th2 type (IgG1) phenotype, were detected in C57BL/6J mice immunized with non-adjuvanted CVB3-VLP vaccine. The structural and immunogenic data presented here indicate the potential of this improved methodology to produce highly immunogenic enterovirus VLP-vaccines in the future. publishedVersion

Viruses need to open, i.e., uncoat, in order to release their genomes for efficient replication and translation. Especially for non-enveloped viruses, such as enteroviruses, the cues leading to uncoating are less well known. The status of the virus has previously been observed mainly by transmission electron microscopy using negative staining, cryo electron microscopy, X-ray crystallography or gradient separation (reviewed in Tuthill et al., 2010, Myllynen et al., 2016, Ruokolainen et al., 2019). However, monitoring of uncoating has been limited by the lack of methods detecting dynamic changes of the virions. Here, we present a real-time fluorescence based protocol, which detects the viral genome (RNA) during various stages of uncoating in vitro, while RNA is still inside the particle that has been expanded before the actual RNA release, and when the RNA has been totally released from the viral particle. Our method allows to explore how various molecular factors may promote or inhibit virus uncoating. peerReviewed

Echovirus 30 (E30), a member of the enterovirus B species, is a major cause of viral meningitis, targeting children and adults alike. While it is a frequently isolated enterovirus and the cause of several outbreaks all over the world, surprisingly little is known regarding its entry and replication strategy within cells. In this study, we used E30 strain Bastianni (E30B) generated from an infectious cDNA clone in order to study early entry events during infection in human RD cells. E30B required the newly discovered Fc echovirus receptor (FcRn) for successful infection, but not the coxsackievirus and adenovirus receptor (CAR) or decay-accelerating factor (DAF), although an interaction with DAF was observed. Double-stranded RNA replication intermediate was generated between 2 and 3 h postinfection (p.i.), and viral capsid production was initiated between 4 and 5 h p.i. The drugs affecting Rac1 (NSC 23766) and cholesterol (filipin III) compromised infection, whereas bafilomycin A1, dyngo, U-73122, wortmannin, and nocodazole did not, suggesting the virus follows an enterovirus-triggered macropinocytic pathway rather than the clathrin pathway. Colocalization with early endosomes and increased infection due to constitutively active Rab5 expression suggests some overlap and entry to classical early endosomes. Taken together, these results suggest that E30B induces an enterovirus entry pathway, leading to uncoating in early endosomes. IMPORTANCE Echovirus 30 (E30) is a prevalent enterovirus causing regular outbreaks in both children and adults in different parts of the world. It is therefore surprising that relatively little is known of its infectious entry pathway. We set out to generate a cDNA clone and gradient purified the virus in order to study the early entry events in human cells. We have recently studied other enterovirus B group viruses, like echovirus 1 (EV1) and coxsackievirus A9 (CVA9), and found many similarities between those viruses, allowing us to define a so-called “enterovirus entry pathway.” Here, E30 is reminiscent of these viruses, for example, by not relying on acidification for infectious entry. However, despite not using the clathrin entry pathway, E30 accumulates in classical early endosomes.

We report that several viruses from the human enterovirus group B cause massive vimentin rearrangements during lytic infection. Comprehensive studies suggested that viral protein synthesis was triggering the vimentin rearrangements. Blocking the host cell vimentin dynamics with IDPN did not significantly affect the production of progeny viruses and only moderately lowered the synthesis of structural proteins such as VP1. In contrast, the synthesis of the non-structural proteins 2A, 3C, and 3D was drastically lowered. This led to attenuation of the cleavage of the host cell substrates PABP and G3BP1 and reduced caspase activation, thus leading to prolonged cell survival. Furthermore, the localization of the proteins differed in the infected cells. Capsid protein VP1 was found diffusely around the cytoplasm, whereas 2A and 3D followed vimentin distribution. Based on protein blotting, lower amounts of non-structural proteins did not result from proteasomal degradation, but from lower synthesis without intact vimentin cage structure. In contrast, inhibition of Hsp90 chaperone activity, which regulates P1 maturation, lowered the amount of VP1, but had less effect on 2A. The results suggest that, the vimentin dynamics regulate viral non-structural protein synthesis while having no effect on structural protein synthesis or overall infection efficiency. The results presented here shed new light on differential fate of structural and non-structural proteins of enteroviruses, having consequences on host cell survival. Importance A virus needs the host cell in order to replicate and produce new progeny viruses. For this, the virus takes over the host cell and modifies it to become a factory for viral proteins. Irrespective of the specific virus family, these proteins can be divided into structural and non-structural proteins. Structural proteins are the building blocks for the new progeny virions, whereas the non-structural proteins orchestrate the take-over of the host cell and its functions. Here we have shown a mechanism that viruses exploit in order to regulate the host cell. We show that viral protein synthesis induces vimentin cages, which promote production of specific viral proteins that eventually control apoptosis and the host cell death. This study specifies vimentin as the key regulator of these events and indicates that viral proteins have different fates in the cells depending on their association with vimentin cages. peerReviewed

A proof-of-concept study shows the generation of an immunogenic hexavalent Coxsackie B virus vaccine that prevents disease., Coxsackievirus B (CVB) enteroviruses are common human pathogens known to cause severe diseases including myocarditis, chronic dilated cardiomyopathy, and aseptic meningitis. CVBs are also hypothesized to be a causal factor in type 1 diabetes. Vaccines against CVBs are not currently available, and here we describe the generation and preclinical testing of a novel hexavalent vaccine targeting the six known CVB serotypes. We show that the vaccine has an excellent safety profile in murine models and nonhuman primates and that it induces strong neutralizing antibody responses to the six serotypes in both species without an adjuvant. We also demonstrate that the vaccine provides immunity against acute CVB infections in mice, including CVB infections known to cause virus-induced myocarditis. In addition, it blocks CVB-induced diabetes in a genetically permissive mouse model. Our preclinical proof-of-concept studies demonstrate the successful generation of a promising hexavalent CVB vaccine with high immunogenicity capable of preventing CVB-induced diseases.

Aims/hypothesis: The Coxsackie and adenovirus receptor (CAR) is a transmembrane cell-adhesion protein that serves as an entry receptor for enteroviruses and may be essential for their ability to infect cells. Since enteroviral infection of beta cells has been implicated as a factor that could contribute to the development of type 1 diabetes, it is often assumed that CAR is displayed on the surface of human beta cells. However, CAR exists as multiple isoforms and it is not known whether all isoforms subserve similar physiological functions. In the present study, we have determined the profile of CAR isoforms present in human beta cells and monitored the subcellular localisation of the principal isoform within the cells. Methods: Formalin-fixed, paraffin-embedded pancreatic sections from non-diabetic individuals and those with type 1 diabetes were studied. Immunohistochemistry, confocal immunofluorescence, electron microscopy and western blotting with isoform-specific antisera were employed to examine the expression and cellular localisation of the five known CAR isoforms. Isoform-specific qRT-PCR and RNA sequencing (RNAseq) were performed on RNA extracted from isolated human islets. Results: An isoform of CAR with a terminal SIV motif and a unique PDZ-binding domain was expressed at high levels in human beta cells at the protein level. A second isoform, CAR-TVV, was also present. Both forms were readily detected by qRT-PCR and RNAseq analysis in isolated human islets. Immunocytochemical studies indicated that CAR-SIV was the principal isoform in islets and was localised mainly within the cytoplasm of beta cells, rather than at the plasma membrane. Within the cells it displayed a punctate pattern of immunolabelling, consistent with its retention within a specific membrane-bound compartment. Co-immunofluorescence analysis revealed significant co-localisation of CAR-SIV with zinc transporter protein 8 (ZnT8), prohormone convertase 1/3 (PC1/3) and insulin, but not proinsulin. This suggests that CAR-SIV may be resident mainly in the membranes of insulin secretory granules. Immunogold labelling and electron microscopic analysis confirmed that CAR-SIV was localised to dense-core (insulin) secretory granules in human islets, whereas no immunolabelling of the protein was detected on the secretory granules of adjacent exocrine cells. Importantly, CAR-SIV was also found to co-localise with protein interacting with C-kinase 1 (PICK1), a protein recently demonstrated to play a role in insulin granule maturation and trafficking. Conclusions/interpretation: The SIV isoform of CAR is abundant in human beta cells and is localised mainly to insulin secretory granules, implying that it may be involved in granule trafficking and maturation. We propose that this subcellular localisation of CAR-SIV contributes to the unique sensitivity of human beta cells to enteroviral infection., SCOPUS: ar.j, info:eu-repo/semantics/published

There is limited information about the molecular triggers leading to the uncoating of enteroviruses under physiological conditions. Using real-time spectroscopy and sucrose gradients with radioactively labeled virus, we show at 37°C, the formation of albumin-triggered, metastable uncoating intermediate of echovirus 1 without receptor engagement. This conversion was blocked by saturating the albumin with fatty acids. High potassium but low sodium and calcium concentrations, mimicking the endosomal environment, also induced the formation of a metastable uncoating intermediate of echovirus 1. Together, these factors boosted the formation of the uncoating intermediate, and the infectivity of this intermediate was retained, as judged by end-point titration. Cryo-electron microscopy reconstruction of the virions treated with albumin and high potassium, low sodium, and low calcium concentrations resulted in a 3.6-Å resolution model revealing a fenestrated capsid showing 4% expansion and loss of the pocket factor, similarly to altered (A) particles described for other enteroviruses. The dimer interface between VP2 molecules was opened, the VP1 N termini disordered and most likely externalized. The RNA was clearly visible, anchored to the capsid. The results presented here suggest that extracellular albumin, partially saturated with fatty acids, likely leads to the formation of the infectious uncoating intermediate prior to the engagement with the cellular receptor. In addition, changes in mono- and divalent cations, likely occurring in endosomes, promote capsid opening and genome release. IMPORTANCE There is limited information about the uncoating of enteroviruses under physiological conditions. Here, we focused on physiologically relevant factors that likely contribute to opening of echovirus 1 and other B-group enteroviruses. By combining biochemical and structural data, we show that, before entering cells, extracellular albumin is capable of priming the virus into a metastable yet infectious intermediate state. The ionic changes that are suggested to occur in endosomes can further contribute to uncoating and promote genome release, once the viral particle is endocytosed. Importantly, we provide a detailed high-resolution structure of a virion after treatment with albumin and a preset ion composition, showing pocket factor release, capsid expansion, and fenestration and the clearly visible genome still anchored to the capsid. This study provides valuable information about the physiological factors that contribute to the opening of B group enteroviruses. peerReviewed

Echoviruses are amongst the most common causative agents of aseptic meningitis worldwide and are particularly devastating in the neonatal population, where they are associated with severe hepatitis, neurological disease, including meningitis and encephalitis, and even death. Here, we identify the neonatal Fc receptor (FcRn) as a pan-echovirus receptor. We show that loss of expression of FcRn or its binding partner beta 2 microglobulin (β2M) renders cells resistant to infection by a panel of echoviruses at the stage of virus attachment, and that a blocking antibody to β2M inhibits echovirus infection in cell lines and in primary human intestinal epithelial cells. We also show that expression of human, but not mouse, FcRn renders nonpermissive human and mouse cells sensitive to echovirus infection and that the extracellular domain of human FcRn directly binds echovirus particles and neutralizes infection. Lastly, we show that neonatal mice expressing human FcRn are more susceptible to echovirus infection by the enteral route. Our findings thus identify FcRn as a pan-echovirus receptor, which may explain the enhanced susceptibility of neonates to echovirus infections.

Type B Coxsackieviruses (CVBs) belong to the enterovirus genus, and they cause both acute and chronic diseases in humans. CVB infections usually lead to flu-like symptoms but can also result in more serious diseases such as myocarditis, aseptic meningitis and life-threatening multi-organ infections in young infants. Thus, CVBs have long been considered as important targets of future vaccines. We have previously observed CVB1 capsid disintegration and virus concentration decrease with 12-day long formalin inactivation protocol. Here a scalable ion exchange chromatography purification method was developed, and purified CVB1 was inactivated with UV-C or formalin. Virus morphology and concentration remained unchanged, when the UV (2 min) or formalin (5 days) inactivation were performed in the presence of tween80 detergent. The concentration of the native and UV inactivated CVB1 remained constant at 4 °C during a six months stability study, whereas the concentration of the formalin inactivated vaccine decreased 29% during this time. UV treatment decreased, whereas formalin treatment increased the thermal stability of the capsid. The formalin inactivated CVB1 vaccine was more immunogenic than the UV inactivated vaccine; the protective neutralizing antibody levels were higher in mice immunized with formalin inactivated vaccine. High levels of CVB1 neutralizing antibodies as well as IgG1 antibodies were detected in mice that were protected against viremia induced by experimental CVB1 infection. In conclusion, this study describes a scalable ion exchange chromatography purification method and optimized 5-day long formalin inactivation method that preserves CVB1 capsid structure and immunogenicity. Formalin treatment stabilizes the virus particle at elevated temperatures, and the formalin inactivated vaccine induces high levels of serum IgG1 antibodies (Th2 type response) and protective levels of neutralizing antibodies. Formalin inactivated CVB vaccines are promising candidates for human clinical trials. peerReviewed

Enteroviruses are small RNA viruses that cause diseases with various symptoms ranging from mild to severe. Enterovirus proteins are translated as a single polyprotein, which is cleaved by viral proteases to release capsid and nonstructural proteins. Here, we show that also cellular calpains have a potential role in the processing of the enteroviral polyprotein. Using purified calpains 1 and 2 in an in vitro assay, we show that addition of calpains leads to an increase in the release of VP1 and VP3 capsid proteins from P1 of enterovirus B species, detected by western blotting. This was prevented with a calpain inhibitor and was dependent on optimal calcium concentration, especially for calpain 2. In addition, calpain cleavage at the VP3-VP1 interface was supported by a competition assay using a peptide containing the VP3-VP1 cleavage site. Moreover, a mass spectrometry analysis showed that calpains can cleave this same peptide at the VP3-VP1 interface, the cutting site being two amino acids aside from 3C&rsquo, s cutting site. Furthermore, we show that calpains cannot cleave between P1 and 2A. In conclusion, we show that cellular proteases, calpains, can cleave structural proteins from enterovirus polyprotein in vitro. Whether they assist polyprotein processing in infected cells remains to be shown.

Enterovirus B species typically cause a rapid cytolytic infection leading to efficient release of progeny viruses. However, they are also capable of persistent infections in tissues, which are suggested to contribute to severe chronic states such as myocardial inflammation and type 1 diabetes. In order to understand the factors contributing to differential infection strategies, we constructed a chimera by combining the capsid proteins from a fast cytolysis causing echovirus 1 (EV1) with non-structural proteins from Coxsackievirus B5 (CVB5) showing persistent infection in RD cells. The results showed that the chimera behaved similar to the parental EV1 leading to efficient cytolysis in both permissive A549 and semi-permissive RD cells. In contrast to EV1 and chimera, CVB5 replicated slower in permissive cells and showed persistent infection in semi-permissive cells. However, there was no difference in the efficiency of uptake of CVB5 in A549 or RD cells in comparison to the chimera or EV1. CVB5 virus batches constantly contained significant amounts of empty capsids, also in comparison to its close relative CVB3. During successive passaging of batch containing only intact CVB5, increasing amounts of empty and decreasing amounts of infective capsids were produced. Our results demonstrate that the increased amounts of empty particles and lowering amounts of infective particles is dictated by the CVB5 structural proteins leading to slowing down the infection between passages. Furthermore, the key factor for persistent infection is the low amount of infective particles produced, not the high number of empty particles accumulating. peerReviewed

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 &minus, RNA and +RNA during acute and persistent enterovirus infections. According to our results, the replication of the viral RNA started 2&ndash, 3 h after infection and the translation shortly after at 3&ndash, 4 h post-infection. The replication hotspots with newly emerging &minus, 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 &minus, RNA and +RNA strands was almost identical, and &minus, 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.

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.

Developments in diagnostic and therapeutic delivery are trending towards molecular level targeting with nano-platforms. Targeted delivery reduces generalized distribution by localizing diagnostic and/or therapeutic (theranostic) molecules to an intended target site. The first section of this thesis proposes Hepatitis E Virus (HEV) nanocapsids as a vector to stabilize and target theranostic delivery. Derived from the capsid protein of HEV, a feco-orally transmitted virus, HEV-like particles self-assemble in to non-infectious, nanocapsids that can withstand harsh protease and pH conditions in the mucosal system. The flexible nanocapsid surface protrusion domain is amenable to substantial modification. Chemical modulation of nanocapsids was achieved through surface conjugation to single solvent exposed cysteine sites. In this thesis, nanocapsids chemically modulated with tumor-targeting ligands exhibit cancer cell-specific binding and internalization, as well as in vivo tumor detection. We also used cysteine sites to conjugate thiolate-protected gold nanoclusters (AuNCs), which have molecule like qualities distinct from colloidal gold nanoparticles (AuNPs). Specifically, Au102(pMBA)44 (Au102) and maleimide-linked Au102 (Au102_C6MI) were conjugated to nanocapsid cysteine through place exchange and maleimide-thiol coupling, respectively. Au102_C6MI-bound nanocapsids were imaged in cryoEM and a 3D structure was determined. The resolved structure of AuNC-bound nanocapsid revealed a 5-fold ring density attributable to AuNC densities. Rigid modelling supported this finding. In the last section of this thesis, a recombinant enterovirus was engineered to distinguish the role of structural vs. non-structural proteins in Enterovirus B infection kinetics, replication and infection persistency. The results indicated cell-receptor binding likely triggered lytic vs. non-lytic infection, providing insight in to adaptive mechanisms of native virus cell delivery.

Enteroviruksilla on suuri merkitys nykymaailmassa. Ne ovat vastuussa sekä suuresta osasta harmittomia tartuntoja että hengenvaarallisista taudeista. Jälkimmäisistä parhaiten tunnettu esimerkki lienee polioviruksen aiheuttama polio. Vaikka rokotteet tarjoavat suojaa poliolta ja joiltain muilta taudeilta, niistä ei ole lopulliseksi ratkaisuksi, sillä ne eivät auta jo tartunnan saaneita ihmisiä. Tätä varten tarvitaan antiviraaleja, yhdisteitä, jotka estävät virusten lisääntymistä. Tutkimus yhdisteiden kehittämiseksi enteroviruksia vastaan on jatkunut jo vuosikymmeniä, mutta ainuttakaan lääkettä ei ole vielä saatu yleiseen hoitokäyttöön. Monia potentiaalisia yhdisteitä on tänä aikana kehitetty ja joitakin testattu kliinisissä kokeissa, mutta niiden suorituskyky ei ole riittänyt koko laajaa taudinaiheuttajaryhmää ja suuresta mutaatioherkkyydestä aiheutuvaa nopeaa resistenttien viruksien kehittymistä vastaan. Tämä pro gradu-tutkielma pyrkii tarjoamaan lyhyen esittelyn enteroviruksista ja syvemmän tarkastelun niitä vastaan kehitetyistä antiviraaleista. Tämä sisältää ensimmäisenä kehitetyt kapsidiin sitoutuvat molekyylit, ei-rakenteellisia viruksen proteiineja vastaan suunnitellut antiviraalit sekä virusinfektiossa hyödynnettyjä solun proteiineja estävät yhdisteet. Yhteenvetokappale pyrkii tarjoamaan ajatuksia kehitystyön tulevaisuudesta ja alati lähenevistä antienteroviraalisista hoidoista. Enteroviruses have a major impact in today’s world. They are responsible for a large portion of harmless infection but also cause life-threatening diseases, poliomyelitis being perhaps the best known example. Although vaccines have proven effective in the abolishment of polio, they cannot be considered as a final solution since they hold no effect on infections that are already taking place. For this, compounds inhibiting the proliferation of the viruses, antivirals, are needed. The research in search for such compounds against enteroviruses has been ongoing for decades, yet no drugs have been put to clinical use so far. Many potent antivirals have been made, a few of them going through clinical trials, but have always proven inefficacious against the diverse range of pathogens and their high mutation rate that provides quick resistance. This work aims to offer an introduction to the enteroviruses and an in depth coverage of many of the antivirals devised against them. This includes the prototype capsid binders, the antivirals created against the non-structural proteins of the viruses as well as the compounds inhibiting the cellular factors involved in the infection found quite recently. The concluding remarks aim to provide some insight to the future of the field and the approaching of antienteroviral treatments.

One of the hallmarks of enterovirus genome delivery is the formation of an uncoating intermediate particle. Based on previous studies of mostly heated picornavirus particles, intermediate particles were shown to have externalized the innermost capsid protein (VP4) and exposed the N terminus of VP1 and to have reduced infectivity. Here, in addition to the native and intact particle type, we have identified another type of infectious echovirus 1 (E1) particle population during infection. Our results show that E1 is slightly altered during entry, which leads to the broadening of the major virion peak in the sucrose gradient. In contrast, CsCl gradient separation revealed that in addition to the light intact and empty particles, a dense particle peak appeared during infection in cells. When the broad peak from the sucrose gradient was subjected to a CsCl gradient, it revealed light and dense particles, further suggesting that the shoulder represents the dense particle. The dense particle was permeable to SYBR green II, it still contained most of its VP4, and it was able to bind to its receptor α 2 β 1 integrin and showed high infectivity. A thermal assay further showed that the α 2 β 1 integrin binding domain (I-domain) stabilized the virus particle. Finally, heating E1 particles to superphysiological temperatures produced more fragile particles with aberrant ultrastructural appearances, suggesting that they are distinct from the dense E1 particles. These results describe a more open and highly infectious E1 particle that is naturally produced during infection and may represent a novel form of an uncoating intermediate. IMPORTANCE In this paper, we have characterized a possible uncoating intermediate particle of E1 that is produced in cells during infection. Before releasing their genome into the host cytosol, enteroviruses go through structural changes in their capsid, forming an uncoating intermediate particle. It was shown previously that structural changes can be induced by receptor interactions and, in addition, by heating the native virion to superphysiological temperatures. Here, we demonstrate that an altered, still infectious E1 particle is found during infection. This particle has a more open structure, and it cannot be formed by heating. It still contains the VP4 protein and is able to bind to its receptor and cause infection. Moreover, we show that in contrast to some other enteroviruses, the receptor-virion interaction has a stabilizing effect on E1. This paper highlights the differences between enterovirus species and further increases our understanding of various uncoating forms of enteroviruses.

Aggresomin muodostuminen on yksi solun puolustusmekanismeista, joka pyrkii estämään proteiinien haitallisen aggregoitumisen solulimassa. Aikaisemmat kokeet ovat näyttäneet, että ihmisen enterovirus-infektio aiheuttaa muutoksia vimentiinissä, tyypin III välikokoisessa filamentissa (Turkki et al., julkaisematon data). Nämä muutokset ovat hyvin samankaltaisia aggresomin muodostumisen aikana tapahtuvien muutoksien kanssa. Tämän tutkimuksen tarkoituksena oli selvittää mahdollisen aggresomin muodostumisreitin aktivoituminen Coxsackievirus B3 (CVB3) -infektiossa, ja hypoteesimme oli, että häkkimäisten vimentiinirakenteiden muodostumisen lisäksi myös muita aggresomin muodostumisreitin aktivoitumiseen yhdistettyjä tapahtumia voidaan havaita CVB3-infektoiduissa soluissa. Lisäksi arvioimme, että aggresomin muodostumisreitin aktivoituminen voi hyödyttää virusinfektiota tai isäntäsolua. Tutkimme työssä näitä aggresomin muodostumiseen liittyviä solumuutoksia infektoiduissa soluissa käyttämällä immunofluoresenssivärjäystekniikoita ja konfokaalimikroskopiaa. Tuloksemme osoittivat, että CVB3-infektio aiheuttaa häkkimäisen vimentiinirakenteen muodostumisen, ja että tämä rakenteellinen muutos vaatii mikrotubuluksia. Infektio aiheuttaa lisäksi suuria rakenteellisia muutoksia solulimakalvostoon ja Golgin laitteeseen, sekä ubikitiinin ja lämpöshokki (Hsp) 70 proteiinin siirtymisen perinukleaariseen tilaan. Tuloksemme viittaavat siihen, että aggresomireitti voi aktivoitua CVB3-infektiossa. Tietojemme mukaan aggresomireitin aktivoitumista ei ole aikaisemmin osoitettu tapahtuvan enterovirusten aiheuttamien infektioiden aikana. Vaikka enteroviruksia on tutkittu kauan, niiden lisääntymiskierron kaikkia eri vaiheita ei tunneta täysin. Tutkimuksemme tulokset tarjoavat uutta tietoa enterovirus-isäntäsolu –vuorovaikutuksista. Aggresome formation is a cellular defence mechanism that is triggered in order to prevent the accumulation of aggregated proteins in the cytoplasm. It has been previously shown that human enterovirus infection causes changes in vimentin, type III intermediate filament protein (Turkki et al., unpublished data), and these structural changes in vimentin resemble the ones induced during aggresome formation. The aim of this study was to investigate the possible activation of aggresomal pathway during Coxsackievirus B3 (CVB3) infection. The hypothesis was that in addition to a vimentin cage, also other factors of the aggresomal pathway might be involved in viral infection, either for the benefit of the virus or host cell. We investigated these cellular changes by using immunofluorescent techniques and confocal microscope imaging. Our results illustrated that CVB3 infection causes a cage-like vimentin, and this rearrangement is dependent on microtubules. In addition, CVB3 infection was shown to induce major structural changes in the ER and Golgi apparatus, accompanied by the translocation of ubiquitin and Heat shock protein (Hsp) 70 to the perinuclear region. These observations indicated that aggresomal pathway could be involved in CVB3 infection. To our best knowledge, the utilization of aggresomal machinery by enteroviruses has not been shown earlier. Despite the fact that enteroviruses have been under research for a long time, there are still areas of the viral lifecycle that are poorly known. Our findings provide more information about enterovirus-host cell –interactions.

Enterovirus B group (EV-B) viruses are important human pathogens which cause a variety of diseases from mild respiratory illnesses to more severe acute infections such as myocarditis and meningitis. EV-Bs have also been associated with chronic infections and autoimmune diseases such as type I diabetes. Because of their significance, better and more accurate methods are necessary to track and visualize viruses in vitro and in vivo. This thesis focus on the development of novel probes to label viruses site-specifically and track infection in vitro. First, we established a covalent conjugation between gold nanocluster markers and EV-B viruses. We were able to visualize through electron microscopy site-specific labelling of cysteines on the viral capsid surface. Second, we developed probes targeted to the hydrophobic pocket of the enterovirus capsid. The probes were synthesized based on a hydrophobic pocket binding drug pleconaril. Successful binding of the probe was confirmed with nuclear magnetic resonance measurements. The probe was conjugated to a fluorescent KU Orange 2 dye or Au102 gold nanocluster, and further studied by confocal and electron microscopy. The third section of this thesis studied the infectious entry pathway of three different coxsackievirus B1 (CVB1) strains. All of the studied strains appeared to use the previously suggested EV-B group entry pathway: macropinocytic entry dependent on Na+/H+ and Rac1 and independent of acidification. However, strain specific kinetic differences in the infection of non- polarized cells were revealed. Importantly, one of the CVB1 strains infected cells independently from endosomal sorting complexes required for transport (ESCRT) driven multivesicular bodies (MVBs), and initiated the virus uncoating with slower kinetics. The results indicate that the ESCRT driven biogenesis of MVBs is required for efficient uncoating and infection. Thus, the viruses show remarkable similarities to other well studied EV-B viruses.

Ihmistä infektoivat enterovirukset luokitellaan ihmisen enterovirus A-, B-, C- ja D -lajeihin sekä rinoviruslajeihin. Nämä lajit sisältävät runsaasti alalajeja, joita tunnetaankin yli 200. Enterovirus on pieni, ikosahedraalinen, genomiltaan yksijuosteinen ja positiivissäikeinen RNA-virus, joka on yleinen taudinaiheuttaja kaikissa ikäryhmissä. Enterovirusinfektioiden epäillään olevan monien autoimmuunisairauksien patogeneesin taustalla. Enteroviruksia pidetään todennäköisimpänä tyypin 1 diabetesta (T1D) aiheuttavana virussukuna, sillä enterovirusinfektioita on todettu diabeetikoilta terveitä useammin monissa eri tutkimuksissa. Vaikka enterovirusinfektiot on yhdistetty T1D:n patogeneesiin, ei enterovirusten osuutta sairauden puhkeamisessa ole kyetty täysin todistamaan. Enterovirusten ja T1D:n välisen epidemiologisen yhteyden osoittaminen on haastavaa, sillä tutkimuksia vaikeuttavat T1D:n oireeton, jopa vuosia kestävä esivaihe sekä haimakudosnäytteiden heikko saatavuus. Vaikka enterovirusdetektiossa immunohistokemialliset menetelmät (IHC) ja in situ -hybridisaatio(ISH) ovat osoittautuneet toimiviksi formaliinifiksattuja kudosnäytteitä käytettäessä, tulee näitä menetelmiä edelleen kehittää detektion luotettavuuden varmistamiseksi. Tutkielman tarkoituksena oli vahvistaa enterovirusdetektion luotettavuutta suunnittelemillamme lajispesifisillä koettimilla sekä omassa laboratoriossamme tuotetuilla polyklonaalisilla enterovirusvasta-aineilla ja arvioida näiden soveltuvuutta enterovirusdetektioon haimakudoksesta. Tutkielman tarkoituksena oli myös selventää tietoa enterovirusten isäntäsoluspesifisyydestä ihmisen haimakudoksessa. Tutkimuksessa näytemateriaalina käytettiin formaliinifiksattuja ja parafiiniin valettuja ihmisten haimakudosleikkeitä ja Balb/c-hiirten monikudosleikkeitä. Enterovirusvastaaineina käytettiin polyklonaalisia kanin vasta-aineita, jotka olivat suunniteltu coxsackievirus A16 (CAV16)-, coxsackievirus B3 (CBV3)- ja echovirus 11 -spesifisiksi. Tutkimuksessa käytettiin ISH-menetelmää sekä kahta erilaista IHC-menetelmää: immunoperoksidaasivärjäystä sekä immunofluoresenssiin perustuvaa kaksoisvärjäystä (IFA). Immunoperoksidaasivärjäyksen avulla seulottiin enteroviruksia ihmisen haimakudosleikkeistä (n=12). Viruspositiivisuutta löydettiin kolmesta haimakudoksesta CAV16 spesifisellä vasta-aineella. CBV3- ja Echo11 -vasta-aineilla positiivisuutta ei löydetty. CAV16 saarekesoluspesifisyyden määrittämiseksi IFA toteutettiin käyttäen CAV16 vasta-ainetta sekä kaupallisia vasta-aineita saarekesoluspesifisiä peptidihormoneja insuliinia, glukagonia ja somatostatiinia vastaan. ISH-määritystä varten enteroviruslajispesifiset DNA-koettimet suunniteltiin sitoutumaan enterovirusgenomin konservoituneille 2C- ja 3D -geenialueille. Lisäksi suunniteltiin koetin, joka sitoutuu enteroviruksen negatiiviseen RNA-juosteeseen, jota virus käyttää templaattina replikaatiossaan. ISH-menetelmä optimoitiin CBV3 infektoidun Balb/c -hiiren kudosleikkeillä. IFA:n mukaan CAV16 infektio rajoittuu haimakudoksessa ainoastaan saarekkeiden insuliinia tuottaviin β-soluihin ja tulokset tukevat yleistä käsitystä enterovirusinfektion β-soluspesifisyydestä. Löytö on merkittävä sillä CAV16 saarekesoluspesifisyyttä ei ole aiemmin osoitettu. Ainoastaan enterovirus B-lajispesifiset koettimet sekä enteroviruksen negatiiviseen juosteeseen sitoutuva koetin, tunnistivat CBV3-virusta hiiren haimakudoksesta, mikä viittaa lajispesifiseen hybridisoitumiseen. Koettimien yhtäaikainen käyttö samassa ISH-reaktiossa ei häirinnyt enterovirus B-lajispesifisten koettimien hybridisoitumista. Itsesuunnitellut koettimet sekä itsetuotetut vasta-aineet lisäävät enterovirusdetektion luotettavuutta merkittävästi, varsinkin jos niitä käytetään kaupallisten koettimien ja vasta-aineiden rinnalla.

Elina Kakkonen tutki väitöskirjassaan kahden erilaisen viruksen, echovirus 1:n ja bakuloviruksen soluun sisäänmenon reittejä ja säätelijöitä. Echovirus 1 (EV1) on ihmisiä infektoiva pikornavirus, joka voi aiheuttaa esimerkiksi aivokalvontulehdusta, sydänlihastulehdusta sekä lievempiä hengitystieinfektioita. Bakulovirus puolestaan on ihmisille vaaraton, hyönteisiä infektoiva virus, joka on osoittautunut lupaavaksi geeninsiirtokuljettajaksi geeniterapiassa.Virukset voivat käyttää soluun tunkeutumisessaan hyväkseen soluissa valmiina olevia erilaisia reittejä, joita solut itse tarvitsevat esimerkiksi ravintoaineiden sisäänottoon. Näin virukset pääsevät solun sisälle solun oman koneiston kuljettamina, siis eräänlaisina vapaamatkustajina. Sekä echovirus 1:n että bakuloviruksen huomattiin käyttävän samankaltaisia reittejä soluun tunkeutumisessaan. Echovirus 1:n kohdalla reitiksi osoittautui makropinosytoosi, ja bakulovirus puolestaan käytti fagosytoosin kaltaista reittiä.- Tulokset olivat erittäin mielenkiintoisia ja yllättäviäkin, sillä näiden kahden reitin ei ole todettu olevan kovinkaan monen viruksen ensisijaisia sisäänmenoreittejä. Lisäksi makropinosytoosin tärkeäksi säätelytekijäksi löysimme proteiinin, jota ei ole aikaisemmin karakterisoitu kyseiselle reitille, Kakkonen kertoo.EV1:n sekä sen reseptorin, α2β1-integriinin, todettiin päätyvän solun sisällä uudenlaisiin rakenteisiin (α2-MVB), jotka eroavat toiminnaltaan ja rakenteeltaan muista solunsisäisistä rakenteista. Lisäksi EV1 ja α2β1-integriini olivat riippuvaisia solukalvon ehjistä kolesterolidomeeneista soluun tunkeutumisessaan. Myös EV1:n kyky infektoida soluja oli riippuvaista ehjistä, kolesterolia sisältävistä α2-MVB-rakenteista, sillä kolesterolin aggregoiminen eli yhteen kokoaminen rakenteiden pinnalla esti virusinfektion tehokkaasti.- Solukalvolla kolesteroli ja sfingolipidit voivat muodostaa hyvin järjestäytyneitä, lateraalisia alueita, ns. lipidilauttoja, joiden merkitystä virusten elinkierron aikana tutkitaan kiivaasti. EV1:n todettiin tarvitsevan kolesterolia ja siten mahdollisesti myös lipidilauttoja sekä soluun tunkeutumisen että viruksen genomin vapautumisen aikana. Myös bakuloviruksen tunkeutuminen soluun alkoi solukalvon kolesterolirikkailta alueilta. Nämä tulokset antavat arvokasta lisätietoa kolesterolin ja lipidilauttojen osallisuudesta virusinfektion etenemisessä, Kakkonen toteaa.Virusten kulkureitit solun sisään on ensin tunnettava, jotta virusten aiheuttamia tauteja voitaisiin estää tai hoitaa. Tulokset auttavat lisäämään ymmärrystämme virusten ja niiden isäntäsolujen monimutkaisista vuorovaikutussuhteista. Lisäksi bakuloviruksen soluun sisäänmenoreitin tunteminen edistää bakulovirusperäisten kuljettimien kehittämistä geeniterapian tarpeisiin To efficiently enter cells, viruses utilize different endocytic pathways. In this thesis, the regulators and entry routes utilized by a human pathogen, echovirus 1 (EV1), as well as an insect pathogen and potential gene therapy vector, baculovirus Autographa californica multiple nucleopolyhedrovirus, were studied. EV1 was shown to enter cells with fluid-phase markers in tubulovesicular structures that matured into multivesicular bodies. The pathway was regulated by factors that have been associated generally with macropinocytosis, namely phospholipase C, phosphatidylinositol 3-kinase, Rac1 and p21-activated kinase-1. C-terminal binding protein1/brefeldin A-ADP ribosylated substrate (CtBP1/BARS) was shown to be a novel regulator of epidermal growth factor-induced macropinocytosis, and CtBP1/BARS also had an essential role in the entry of EV1 and α2β1 integrin, the receptor of EV1. EV1 and α2β1 integrin originated from lipid raft-derived plasma membrane areas, and the internalization was dependent on cholesterol both at the plasma membrane and during the later stages of entry. The multivesicular structures formed after α2β1 integrin clustering (α2-MVBs) were sensitive to cholesterol-aggregating drugs, but not to Triton X-100 treatment, indicating that they were enriched in cholesterol and possibly raft lipids. Cholesterol-aggregating drugs also inhibited EV1 uncoating and had a great impact in the cytoplasmic α2-MVB structures.Baculovirus internalization into mammalian cells was sensitive to a cholesterol-aggregating drug, and the internalization induced membrane ruffling. The virus entered cells together with fluid-phase markers in large, noncoated vesicles. Regulators of the clathrin-mediated pathway or macropinocytosis were not involved in baculovirus entry. Instead, the pathway was regulated by RhoA and Arf6, and the virus triggered the uptake of phagocytic marker E.coli into non-phagocytic human cells. These results suggest that baculovirus entry occurs via a mechanism that is reminiscent of phagocytosis