Browsing by Subject " virology"

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  • Pietilä, Maija (Helsingin yliopisto, 2013)
    Extremophiles are found in all three domains of cellular life but especially archaea are able to withstand harsh conditions. Halophilic archaea thrive in hypersaline environments like salt lakes and salterns which have been shown to contain high abundance of virus-like particles. So far, head-tailed viruses are the most common isolates infecting haloarchaea, which is in contrast to a variety of morphologies described for the viruses of hyper-thermophilic archaea. Altogether, approximately 100 archaeal viruses have been isolated but only a fraction of them has been subjected to detailed structural analyses. In this thesis, a novel haloarchaeal virus, Halorubrum pleomorphic virus 1 (HRPV-1), was isolated from a solar saltern. This virus was shown to have a flexible, pleomorphic vesicle-like virion devoid of a rigid protein capsid. The genome analyses revealed that HRPV-1 is the first archaeal virus to be isolated which does not have a double-stranded but a single-stranded DNA genome. A genomic region of HRPV-1 showed similarity to the genome of another haloarchaeal virus, Haloarcula hispanica virus 2 (His2), as well as to the genome of Haloarcula marismortui and Natronomonas pharaonis indicating that HRPV-1-like elements are widespread. Consistent with this, pleomorphic viruses resembling HRPV-1 and infecting haloarchaea of the genera Haloarcula, Halorubrum and Halogeometricum have subsequently been isolated from geographically distant locations, and this study was extended to altogether seven viruses. All these viruses were sensitive to lowered ionic strength confirming their halophilic nature. Based on the virion properties, these haloviruses were defined as pleolipoviruses. Life-cycle studies showed that the pleolipoviruses are nonlytic and progeny virions are produced continuously resulting in host growth retardation. The most likely exit mechanism is budding which is consistent with the observation that the pleolipoviruses acquire their lipids unselectively from the host lipid pool. All pleolipoviruses have two major structural protein species, and biochemical dissociation studies showed that the larger-sized proteins form spike-like protrusions on the virion surface and the smaller-sized proteins are embedded in the inner surface of the membrane vesicle. The three-dimensional virion structure of HRPV-1 revealed that the spike structures are randomly distributed on the virion surface. The genome of the pleolipoviruses is enclosed in a lipid vesicle without associated nucleoproteins. Although the pleolipoviruses have different genome types, single- or double-stranded, circular or linear DNA, the membrane vesicle-based virion architecture is conserved. This work introduced a novel group of pleomorphic viruses infecting extremely halophilic archaea and showed that vesicle-like virion architecture is common in hypersaline environments. Interestingly, the archaeal pleolipoviruses were observed to share several similarities with a bacterial mycoplasmavirus indicating that these viruses may form a viral lineage with an ancient origin.
  • Hetzel, Udo; Szirovicza, Leonora; Smura, Teemu; Prahauser, Barbara; Vapalahti, Olli; Kipar, Anja; Hepojoki, Jussi (2019)
    Hepatitis D virus (HDV) forms the genus Deltavirus unassigned to any virus family. HDV is a satellite virus and needs hepatitis B virus (HBV) to make infectious particles. Deltaviruses are thought to have evolved in humans, since for a long time, they had not been identified elsewhere. Herein we report, prompted by the recent discovery of an HDV-like agent in birds, the identification of a deltavirus in snakes (Boa constrictor) designated snake HDV (sHDV). The circular 1,711-nt RNA genome of sHDV resembles human HDV (hHDV) in its coding strategy and size. We discovered sHDV during a metatranscriptomic study of brain samples of a Boa constrictor breeding pair with central nervous system signs. Applying next-generation sequencing (NGS) to brain, blood, and liver samples from both snakes, we did not find reads matching hepadnaviruses. Sequence comparison showed the snake delta antigen (sHDAg) to be 55% and 37% identical to its human and avian counterparts. Antiserum raised against recombinant sHDAg was used in immunohistology and demonstrated a broad viral target cell spectrum, including neurons, epithelial cells, and leukocytes. Using RT-PCR, we also detected sHDV RNA in two juvenile offspring and in a water python (Liasis mackloti savuensis) in the same snake colony, potentially indicating vertical and horizontal transmission. Screening of 20 randomly selected boas from another breeder by RT-PCR revealed sHDV infection in three additional snakes. The observed broad tissue tropism and the failure to detect accompanying hepadnavirus suggest that sHDV could be a satellite virus of a currently unknown enveloped virus. IMPORTANCE So far, the only known example of deltaviruses is the hepatitis delta virus (HDV). HDV is speculated to have evolved in humans, since deltaviruses were until very recently found only in humans. Using a metatranscriptomic sequencing approach, we found a circular RNA, which resembles that of HDV in size and coding strategy, in a snake. The identification of similar deltaviruses in distantly related species other than humans indicates that the previously suggested hypotheses on the origins of deltaviruses need to be updated. It is still possible that the ancestor of deltaviruses emerged from cellular RNAs; however, it likely would have happened much earlier in evolution than previously thought. These findings open up completely new avenues in evolution and pathogenesis studies of deltaviruses.
  • Anastasina, Maria (Helsingin yliopisto, 2015)
    Viruses infect all domains of life. They establish complex interactions with their host cells to subvert and hijack multiple cellular processes and warrant their own replication. Understanding virus-host interactions is critical to control spread of pathogenic viruses, develop vaccines and search for antivirals. Besides that, understanding virus-host interactions allows deciphering complex cellular processes and provides useful tools for biotechnology. My research is dedicated to influenza A virus, an important pathogen that infects humans worldwide, represents a constant health care threat and elicits continuous efforts to control the human spread of the disease. Influenza A expresses a non-structural protein NS1 that is a key regulator of viral interactions with the host cell and an important virulence factor. Versatile functions of NS1 modulate multiple cellular functions to secure viral replication. This work addresses several aspects of NS1-mediated modulation of core cellular processes. We discovered that NS1 binds to dsDNA and inhibits transcription of cellular genes, thus limiting antiviral responses. We found that NS1 secures general protein synthesis and mapped several residues within NS1 that are essential for this function. Further, we showed that the length of C-terminal ``tail'' of NS1 is essential for control of cellular antiviral responses and virus pathogenicity. The presented results increase the understanding of influenza A virus-host interactions and can be further utilized in the search for antivirals and vaccine development. In addition, this work provides a biotechnological application of influenza A NS1 protein for improvement of cell-free translation system.
  • Denisova, Oxana (Helsingin yliopisto, 2014)
    Influenza viruses cause pandemics and annual epidemics which have serious consequences for public health and global economy. The severity of infections with influenza viruses can vary from asymptomatic to life-threatening viral pneumonias. Currently, four licensed anti-influenza drugs are available for the prevention and treatment of influenza virus infections. However, resistance to the licensed antivirals develops rapidly. Therefore, there is a need for next-generation antiviral agents to combat influenza virus infections. Recent advances in understanding influenza virus-host interactions have revealed a number of host targets for potential antiviral interventions. In particular, basic cellular functions, metabolic and biosynthesis pathways, as well as the signaling cascades could be modulated by small-molecule inhibitors to block virus infection. Moreover, temporal inhibition of these host functions will be less likely to induce viral drug resistance. In addition, many of the inhibitors of cellular functions are already approved or in clinical development for other diseases. Drug repurposing will facilitate their introduction for treatment of viral infections, since the pharmacokinetics and toxicity profile of these drugs are already known. In this work, a library of small-molecule inhibitors targeting host factors and potentially interfering with influenza virus infection was built and screened. Inhibitors of vacuolar proton-ATPase (v-ATPase), Akt kinase, ribonucleotide reductase and the anti-apoptotic B-cell lymphoma-2 family proteins showed antiviral activity in vitro. Saliphenylhalamide, an inhibitor of v-ATPase, was the most potent antiviral agent and it was effective against a broad range of influenza viruses and some other RNA viruses in vitro, and against a mouse adapted influenza strain in vivo. In order to overcome the low water solubility and high toxicity of saliphenylhalamide, bioavailability was optimized using a porous silicon particle-based delivery system for the putative clinical trials. The results presented in this study expand the understanding of influenza virus-host interactions, and provide a novel perspective for ways to adopt a rational approach in the discovery of new antiviral agents.
  • Barenkamp, Stephen J.; Chonmaitree, Tasnee; Hakansson, Anders P.; Heikkinen, Terho; King, Samantha; Nokso-Koivisto, Johanna; Novotny, Laura A.; Patel, Janak A.; Pettigrew, Melinda; Swords, W. Edward (2017)
    Objective. To perform a comprehensive review of the literature from July 2011 until June 2015 on the virology and bacteriology of otitis media in children. Data Sources. PubMed database of the National Library of Medicine. Review Methods. Two subpanels comprising experts in the virology and bacteriology of otitis media were created. Each panel reviewed the relevant literature in the fields of virology and bacteriology and generated draft reviews. These initial reviews were distributed to all panel members prior to meeting together at the Post-symposium Research Conference of the 18th International Symposium on Recent Advances in Otitis Media, National Harbor, Maryland, in June 2015. A final draft was created, circulated, and approved by all panel members. Conclusions. Excellent progress has been made in the past 4 years in advancing our understanding of the microbiology of otitis media. Numerous advances were made in basic laboratory studies, in animal models of otitis media, in better understanding the epidemiology of disease, and in clinical practice. Implications for Practice. (1) Many viruses cause acute otitis media without bacterial coinfection, and such cases do not require antibiotic treatment. (2) When respiratory syncytial virus, metapneumovirus, and influenza virus peak in the community, practitioners can expect to see an increase in clinical otitis media cases. (3) Biomarkers that predict which children with upper respiratory tract infections will develop otitis media may be available in the future. (4) Compounds that target newly identified bacterial virulence determinants may be available as future treatment options for children with otitis media.
  • Korzyukov, Yegor (Helsingin yliopisto, 2020)
    The family Arenaviridae is a well-represented clade of RNA viruses. The genus Mammarenavirus is dominated by rodent-borne arenaviruses, several of which have been identified as the causative agents behind hemorrhagic fevers and neurological infections in humans. Despite having been studied for more than 90 years, mammarenavirus diagnostics, vaccines and antiviral compounds are only available for some mammarenaviruses. Another genus, Reptarenavirus, includes viruses linked to boid inclusion body disease (BIBD) in constrictor snakes. BIBD has been reported in captive constrictor snake species since the 1970s, but the etiological agents were only identified in 2012. BIBD can lead to the eradication of the entire affected snake populations. The range of possible host spectrum and the immune response against reptarenaviruses are not well characterized. This thesis aims to define the potential reptarenavirus host cell spectrum as well as expand understanding of the boid immune response. One of the hallmark signs of reptarenavirus infection in snakes is the formation of inclusion bodies (IB) in host cells. Snakes are poikilotherm and the replication of viruses is often susceptible to temperature variation. Reptarenavirus infection in mammalian, boid, and arthropod cells, incubated at 37 °C did not induce IB formation, whereas prominent IB formation occurred in all three phyla when incubated at 30°C. Reptarenaviruses replicated efficiently at 30°C, whereas at 37°C the replication efficiency reduced significantly. Many animal viruses take advantage of glycoproteins (GPs) to mediate binding and entry via attachment to host cell surface receptors. To study the ability of reptarenavirus GPs to mediate cell entry, a pseudovirus system based on reporter gene-bearing recombinant vesicular stomatitis virus (rVSV) was introduced. The pseudoviruses with reptarenavirus GPs served to demonstrate that the majority of arenavirus GPs could mediate entry to both mammalian and reptilian cells but at varying efficiencies. In order to validate the link between BIBD and reptarenavirus infection, constrictor snakes (Boa constrictor and Python regius) were experimentally infected. Despite transient central nervous system signs, IB were not detected in the infected snakes. The snakes were sacrificed and sera was collected to determine the magnitude of the humoral immune response. In order to assess the antibody response against reptarenaviruses it was necessary to develop reagents capable of detecting immunoglobulins in snake sera. The generated reagents were initially tested using sera from BIBD-positive snakes. IgY and IgM class antibodies binding reptarenaviruses were detected in serum samples, validating the functionality of the reagents. In the next study, these antibodies were used to detect IgM and IgY antibodies in experimentally and naturally infected snake populations. Extracted sera was further assayed using the rVSV-based pseudoviruses decorated with reptarenavirus GPs to show a neutralizing antibody response following reptarenavirus infection. This thesis adds to understanding of reptarenavirus infectivity across species barriers. The generation of novel diagnostic reagents will allow generation of serodiagnostic tools for reptarenavirus infection. Future studies of reptarenaviruses should aim to establish a virus-specific link between reptarenaviruses and BIBD that could serve in the development of effective and preventive treatment strategies.
  • Chen, Tingting (Helsingin yliopisto, 2013)
    Two newly found polyomaviruses are related to human skin diseases; Merkel cell polyomavirus (MCPyV), identified in Merkel cell carcinomas (MCC) is believed to be an etiologic factor in the pathogenesis of MCC; trichodysplasia spinulosa-associated polyomavirus (TSPyV), discovered in TS lesions, has an apparently causative role in this rare skin disease. To investigate their seroprevalence and possible clinical significance, we have developed for these two polyomaviruses comprehensive serodiagnostic methods and studied the occurrence of antibodies to these viruses, the quality of immune responses, diagnostic criteria, and possible disease associations. In addition, the thesis aims at searching for factors that cause false results in biotinylation-based antiviral IgM enzyme immunoassays (EIAs). For MCPyV and TSPyV, we developed EIAs for virus-specific IgG, IgM and IgG avidity, by using the major capsid protein VP1 expressed in insect cells as self-assembled into virus-like particles. To determine seroprevalence and seroconversion rates, virus-specific IgG antibody assays were applied to examine serum samples from adults and three groups of children less than 13 years of age. In the first two children´s groups, children underwent initial and 3-8-year follow-up sampling. In the third group, children were followed clinically from birth until 13 years of age, and from each such child consecutive serum samples were obtained. Among Finnish adults, the seroprevalence for MCPyV was 58% and for TSPyV 70%. Among Finnish children between 1-13 years of age, the seroprevalence for MCPyV were determined at 9-45%, with seroconversion rates of 16-33% during the 3-8-year follow-up, while for TSPyV the seroprevalence was 5-39%, with seroconversion rates of 30-45%. Among 144 children followed from birth up to 13 years, 31% and 27% had IgG seroconversion to MCPyV and to TSPyV, respectively. These data indicate that MCPyV and TSPyV circulate widely in the general population, and that primary infections by these two viruses occur extensively in childhood with the first exposures taking place at young age. We then investigated whether in children primary infections with these two viruses were associated with any symptoms or signs. For each virus, EIAs for antiviral IgG, IgM and IgG avidity, along with quantitative PCR were employed on consecutive serum samples from the seroconverting children in the third children´s group. At the time of IgG seroconversion, additional infection markers (IgM response and/or low-avidity IgG) were found to be present in 62% of the MCPyV seroconverters, and in 82% of the TSPyV seroconverters. Comparing clinical events during the seroconversion intervals with those during the previous and subsequent intervals, we found that with neither virus primary infection was significantly associated with symptoms or signs of any infection-related illness. These observations imply that in childhood primary infections with MCPyV or TSPyV are in general asymptomatic. To improve the quality of serodiagnostic assays, we looked for factors causing false IgM reactivities in IgM assays in which captured virus-specific IgM antibodies are detected by biotinylated viral antigens bound to horseradish peroxidase conjugated streptavidin. Employed as negative control for our MCPyV IgM EIA, one adult serum sample exhibited IgM reactivity against the biotinylated viral antigen, but did not show reactivity against the non-biotinylated one. This observation pointed to the occurrence of anti-biotin IgM in human blood, causing false-positive results in biotinylation-based antiviral IgM assays. To investigate the prevalence of biotin-specific IgM antibodies in the general population, we developed an indirect EIA for biotin IgM, and found these antibodies to be present in 3% of adults regardless of age but rarely in children. Adverse effects of the biotin IgM were examined with a variety of biotinylation-based antiviral IgM assays. Interestingly, soluble biotin failed to bind the IgM and streptavidin/avidin simultaneously, pointing to the possibility that the IgM and streptavidin/avidin share the same binding site on biotin. The affinities of biotin IgM antibodies were determined at a range of 10-3 to 10-4 mol/L. These findings provide new information on biotinylation-based immunoassays, and open new insights into anti-vitamin immune responses.
  • Jääskeläinen, Anne J; Voutilainen, Liina; Lehmusto, R.; Henttonen, H.; Lappalainen, Maija; Kallio-Kokko, H.; Vaheri, A.; Vapalahti, O. (2016)
    Ljungan virus (LV) is a picornavirus related to human parechoviruses (HPeV). The virus has been found in bank voles (Myodes glareolus) and several other rodent species, and suggested to have zoonotic potential. Thus far, seroepidemiological data on LV infections in humans are scarce. In this study, we aimed to characterize the demographic and geographical distribution of LV-reactive antibodies in Finland, and to investigate its occurrence in patients suspected of having a rodent-borne disease, nephropathia epidemica (NE) caused by Puumala hantavirus (PUUV). Using an immunofluorescence assay (LV strain 145SLG), we screened human sera (n = 1378) and found LV-reactive antibodies in 36% of samples. The probability of possessing LV-reactive antibodies peaked at age of 14 years, suggesting that most infections occur in childhood. The prevalence of LV-reactive antibodies was significantly higher in the urbanized area surrounding Helsinki than in more rural Central Finland. These findings are uncharacteristic of a rodent-borne pathogen, and therefore we consider human-to-human transmission of one or several Ljungan-like viruses as a likely cause for most of the observed antibody responses.
  • Szirovicza, Leonora; Hetzel, Udo; Kipar, Anja; Martinez-Sobrido, Luis; Vapalahti, Olli; Hepojoki, Jussi (2020)
    Satellite viruses, most commonly found in plants, rely on helper viruses to complete their replication cycle. The only known example of a human satellite virus is the hepatitis D virus (HDV), and it is generally thought to require hepatitis B virus (HBV) to form infectious particlee03250-19s. Until 2018, HDV was the sole representative of the genus Deltavirus and was thought to have evolved in humans, the only known HDV host. The subsequent identification of HDV-like agents in birds, snakes, fish, amphibians, and invertebrates indicated that the evolutionary history of deltaviruses is likely much longer than previously hypothesized. Interestingly, none of the HDV-like agents were found in coinfection with an HBV-like agent, suggesting that these viruses use different helper virus(es). Here we show, using snake deltavirus (SDeV), that HBV and hepadnaviruses represent only one example of helper viruses for deltaviruses. We cloned the SDeV genome into a mammalian expression plasmid, and by transfection could initiate SDeV replication in cultured snake and mammalian cell lines. By superinfecting persistently SDeV-infected cells with reptarenaviruses and hartmaniviruses, or by transfecting their surface proteins, we could induce production of infectious SDeV particles. Our findings indicate that deltaviruses can likely use a multitude of helper viruses or even viral glycoproteins to form infectious particles. This suggests that persistent infections, such as those caused by arenaviruses and orthohantaviruses used in this study, and recurrent infections would be beneficial for the spread of deltaviruses. It seems plausible that further human or animal disease associations with deltavirus infections will be identified in the future.IMPORTANCE Deltaviruses need a coinfecting enveloped virus to produce infectious particles necessary for transmission to a new host. Hepatitis D virus (HDV), the only known deltavirus until 2018, has been found only in humans, and its coinfection with hepatitis B virus (HBV) is linked with fulminant hepatitis. The recent discovery of deltaviruses without a coinfecting HBV-like agent in several different taxa suggested that deltaviruses could employ coinfection by other enveloped viruses to complete their life cycle. In this report, we show that snake deltavirus (SDeV) efficiently utilizes coinfecting reptarena- and hartmaniviruses to form infectious particles. Furthermore, we demonstrate that cells expressing the envelope proteins of arenaviruses and orthohantaviruses produce infectious SDeV particles. As the envelope proteins are responsible for binding and infecting new host cells, our findings indicate that deltaviruses are likely not restricted in their tissue tropism, implying that they could be linked to animal or human diseases other than hepatitis.
  • Karelehto, Eveliina; van der Sanden, Sabine; Geraets, James A.; Domanska, Ausra; van der Linden, Lonneke; Hoogendoorn, Dionne; Koen, Gerrit; van Eijk, Hetty; Shakeel, Shabih; Beaumont, Tim; de Jong, Menno; Pajkrt, Dasja; Butcher, Sarah J.; Wolthers, Katja C. (2017)
    Human parechovirus 3 (HPeV3), a member of the Picornavirus family, is frequently detected worldwide. However, the observed seropositivity rates for HPeV3 neutralizing antibodies (nAbs) vary from high in Japan to low in the Netherlands and Finland. To study if this can be explained by technical differences or antigenic diversity among HPeV3 strains included in the serological studies, we determined the neutralizing activity of Japanese and Dutch intravenous immunoglobulin batches (IVIG), a rabbit HPeV3 hyperimmune polyclonal serum, and a human HPeV3-specific monoclonal antibody (mAb) AT12-015, against the HPeV3 A308/99 prototype strain and clinical isolates from Japan, the Netherlands and Australia, collected between 1989 and 2015. The rabbit antiserum neutralized all HPeV3 isolates whereas the neutralization capacity of the IVIG batches varied, and the mAb exclusively neutralized the A308/99 strain. Mapping of the amino acid variation among a subset of the HPeV3 strains on an HPeV3 capsid structure revealed that the majority of the surface-exposed amino acid variation was located in the VP1. Furthermore, amino acid mutations in a mAb AT12-015-resistant HPeV3 A308/99 variant indicated the location for potential antigenic determinants. Virus aggregation and the observed antigenic diversity in HPeV3 can explain the varying levels of nAb seropositivity reported in previous studies.