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Infection, early entry events and replication processes of picornaviruses
Linnaeus University, Faculty of Health and Life Sciences, Department of Chemistry and Biomedical Sciences.
2020 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The Picornaviridae is a large and diverse family of small RNA viruses, several of which have exhibited a distinct clinical and socioeconomic impact on human society. While many infections are asymptomatic, some members of this virus family have the potential to cause severe disease, including, but not limited to, poliomyelitis, myocarditis, meningitis, and foot-and-mouth disease. Picornaviruses have been the knowledge foundation of modern virology, and as such, they have been studied extensively over the past century. This thesis aimed to further that understanding by examining the early entry and replication kinetics of echovirus 30 (E30), coxsackievirus B5 (CVB5), and rhinovirus C34 (RV-C34), as well as the presence and transmission of Saffold virus (SAFV) in Sweden. 

E30 appears to exploit one previously known enterovirus entry pathway to facilitate its infection, using DAF as an attachment receptor and leading to early endosomal uncoating. In CVB5, it is the structural proteins that dictate the increase in empty particles and reduction in infective particles, resulting in a slowed infection between passages in cultured cells. As it stands, the key factor to establish a persistent infection seems to be the small number of infective particles as opposed to the high number of empty particles. Furthermore, we present in this thesis the prototype genome of RV-C34, a member of the rhinovirus C species which shows limited replication potential in cell culture. Finally, analysis of Swedish patient samples confirmed the circulation of SAFV-3 in Sweden and, for the first time, demonstrated the presence of this virus in elderly people. Furthermore, we showed that the circulating strain is phylogenetically closely related to several Asian strains, as well as the Dutch strain described in 2009. 

Taken together, this thesis contributes to a better understanding of enteroviral entry and infection, as well as SAFV epidemiology in Sweden. 

Place, publisher, year, edition, pages
Växjö: Linnaeus University Press, 2020. , p. 70
Series
Linnaeus University Dissertations ; 378/2020
Keywords [en]
Picornavirus, enterovirus, cardiovirus, echovirus, rhinovirus, coxsackievirus B, Saffold virus, early entry, replication, gastroenteritis, aseptic meningitis.
National Category
Microbiology
Research subject
Biomedical Sciences, Virology
Identifiers
URN: urn:nbn:se:lnu:diva-95674Libris ID: 8lzkj6t8666k1vlcISBN: 978-91-89081-40-6 (print)ISBN: 978-91-89081-41-3 (electronic)OAI: oai:DiVA.org:lnu-95674DiVA, id: diva2:1436204
Public defence
2020-06-16, Vi2166 (Azur), Norra Kajplanen 6, Kalmar, 09:00 (English)
Opponent
Supervisors
Available from: 2020-06-08 Created: 2020-06-07 Last updated: 2024-02-29Bibliographically approved
List of papers
1. Early Entry Events in Echovirus 30 Infection
Open this publication in new window or tab >>Early Entry Events in Echovirus 30 Infection
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2020 (English)In: Journal of Virology, ISSN 0022-538X, E-ISSN 1098-5514, Vol. 94, no 13, p. 1-15, article id e00592-20Article in journal (Refereed) Published
Abstract [en]

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.

Place, publisher, year, edition, pages
American Society of Microbiology, 2020
Keywords
aseptic meningitis, early entry, echovirus 30, enterovirus
National Category
Microbiology
Research subject
Biomedical Sciences, Virology
Identifiers
urn:nbn:se:lnu:diva-97723 (URN)10.1128/JVI.00592-20 (DOI)000550171000009 ()32295914 (PubMedID)2-s2.0-85086682908 (Scopus ID)
Available from: 2020-08-27 Created: 2020-08-27 Last updated: 2021-05-07Bibliographically approved
2. Slow Infection due to Lowering the Amount of Intact versus Empty Particles Is a Characteristic Feature of Coxsackievirus B5 Dictated by the Structural Proteins
Open this publication in new window or tab >>Slow Infection due to Lowering the Amount of Intact versus Empty Particles Is a Characteristic Feature of Coxsackievirus B5 Dictated by the Structural Proteins
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2019 (English)In: Journal of Virology, ISSN 0022-538X, E-ISSN 1098-5514, Vol. 93, no 20, p. 1-15, article id e01130-19Article in journal (Refereed) Published
Abstract [en]

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 fast-cytolysis-causing echovirus 1 (EV1) with nonstructural proteins from coxsackievirus B5 (CVB5), which shows persistent infection in RD cells. The results showed that the chimera behaved similarly to parental EV1, leading to efficient cytolysis in both permissive A549 and semipermissive RD cells. In contrast to EV1 and the chimera, CVB5 replicated slowly in permissive cells and showed persistent infection in semipermissive 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 batches constantly contained significant amounts of empty capsids, also in comparison to CVBS's close relative CVB3. During successive passaging of batches containing only intact CVB5, increasing amounts of empty and decreasing amounts of infective capsids were produced. Our results demonstrate that the increase in the amount of empty particles and the lowering of the amount of infective particles are dictated by the CVB5 structural proteins, leading to slowing down of the infection between passages. Furthermore, the key factor for persistent infection is the small amount of infective particles produced, not the high number of empty particles that accumulate. IMPORTANCE Enteroviruses cause several severe diseases, with lytic infections that lead to rapid cell death but also persistent infections that are more silent and lead to chronic states of infection. Our study compared a cytolytic echovirus 1 infection to persistent coxsackievirus B5 infection by making a chimera with the structural proteins of echovirus 1 and the nonstructural proteins of coxsackievirus B5. Coxsackievirus 85 infection was found to lead to the production of a high number of empty viruses (empty capsids) that do not contain genetic material and are unable to continue the infection. Coinciding with the high number of empty capsids, the amount of infective virions decreased. This characteristic property was not observed in the constructed chimera virus, suggesting that structural proteins are in charge of these phenomena. These results shed light on the mechanisms that may cause persistent infections. Understanding events leading to efficient or inefficient infections is essential in understanding virus-caused pathologies.

Place, publisher, year, edition, pages
American Society of Microbiology, 2019
Keywords
enterovirus, infection kinetics, viral nonstructural proteins, viral structural proteins, virus-host interactions
National Category
Microbiology
Research subject
Ecology, Microbiology
Identifiers
urn:nbn:se:lnu:diva-89707 (URN)10.1128/JVI.01130-19 (DOI)000488281200032 ()31375587 (PubMedID)2-s2.0-85072791952 (Scopus ID)
Available from: 2019-10-18 Created: 2019-10-18 Last updated: 2020-12-14Bibliographically approved
3. Genetic analysis and limited replication properties of therhinovirus C34 prototype
Open this publication in new window or tab >>Genetic analysis and limited replication properties of therhinovirus C34 prototype
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(English)Manuscript (preprint) (Other academic)
National Category
Microbiology
Research subject
Biomedical Sciences, Virology
Identifiers
urn:nbn:se:lnu:diva-97748 (URN)
Available from: 2020-08-27 Created: 2020-08-27 Last updated: 2020-08-27Bibliographically approved
4. Saffold virus infection in elderly people with acutegastroenteritis in Sweden
Open this publication in new window or tab >>Saffold virus infection in elderly people with acutegastroenteritis in Sweden
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(English)Manuscript (preprint) (Other academic)
National Category
Microbiology
Research subject
Biomedical Sciences, Virology
Identifiers
urn:nbn:se:lnu:diva-97749 (URN)
Available from: 2020-08-27 Created: 2020-08-27 Last updated: 2020-08-27Bibliographically approved

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