Browsing by Subject "virologia"

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  • Wang, Hongjie (Helsingin yliopisto, 2012)
    Human adenoviruses (Ads) have been classified into six species (A to F) currently containing 55 serotypes. For almost 2 decades vectors derived from group C serotype Ad5 have been extensively used for gene transfer studies. These Ad5 based vectors are able to efficiently infect many mammalian cell types (including both mitotic and post-mitotic cells) through interaction with a primary attachment receptor, the coxsackie and adenovirus receptor (CAR). Despite the many advantages of Ad5 based vectors a number of limitations have affected their therapeutic application to many diseases. Although they can transduce many tissue types, Ad5 based vectors are unable to efficiently transduce several potential disease target cell types, including hematopoietic stem cells and malignant tumor cells. Therefore, newer vectors have been developed based on Ad serotypes other than Ad5. This thesis focuses on species B Ads. Species B Ads are comprised of three groups based on their receptor usage. Group 1 of species B Ads (Ad16, 21, 35, 50) nearly exclusively utilize CD46 as a receptor; Group 2 (Ad3, Ad7, 14) share a common, unidentified receptor/s, which is not CD46 and which was tentatively named receptor X; Group 3 (Ad11) preferentially interacts with CD46, but also utilizes receptor X if CD46 is blocked. Species B group Ads are important human pathogens. Species B group 2 serotypes are isolated from patients with respiratory tract infections, whereas the Group 1 viruses are described as causing kidney and urinary tract infections. B-group Ad infections often occur in immunocompromised patients, including AIDS patients, recipients of bone marrow transplants, or chemotherapy patients. Recent studies performed in U.S. military training facilities indicate an emergence of diverse species B serotypes at the majority of sites. This included the group 1 serotype 21 and the group 2 serotypes 3, 7, and 14. CD46-targeting vectors derived from Ad35 and Ad11 are important tools for in vitro gene transfer into human stem cells, including hematopoietic stem cells and induced pluripotent stem cells. Ad35 and Ad11 have been used as tools for cancer therapy, because CD46 appears to be uniformely overexpressed on many cancers. Furthermore, receptor X-targeting vectors, i.e vectors derived from Ad3 or vectors containing Ad3 fibers have shown superior in the transduction of tumor cells both in vitro and in vivo and are currently being used clinically in cancer patients. While extensive basic virology studies have been done on Ad5, the information of species B group 1 interaction with CD46 is limited. Furthermore, the receptor for a major subgroup of species B Ads (receptor X) is unknown. The goal of this thesis was it therefore to better understand virological and translational aspects of species B Ads. The specific findings described in this thesis include i) the identification of CD46 binding sites within the Ad35 fiber knob, ii) the study of the in vitro and in vivo properties of Ad vectors with increased affinity to CD46. iii) the study of the receptor usage of a newly emergent Ad14a, iv) the identification of desmoglein 2 as the receptor for Ad3, Ad7, Ad11, and Ad14, v) the delineation of structural details of Ad3 virus interaction with DSG2, and vi) the analysis of functional consequences of Ad3-DSG2 interaction. As a result of these basic virology studies two Ad-derived recombinant proteins have been generated that can be used to enhance cancer therapy by monoclonal antibodies.
  • Putkuri, Niina (Helsingin yliopisto, 2016)
    Inkoo virus (INKV) is a mosquito-borne virus belonging to the California serogroup (genus Orthobunyavirus, family Bunyaviridae) which includes many important human pathogens described especially in the USA. The association of INKV infection with clinical disease has not been confirmed, but occasional cases of meningitis and encephalitis have been diagnosed. However, the true incidence of acute infections is not known because ongoing research and laboratory diagnostics of INKV infections have been neglected for decades in the countries where the virus circulates. We established a serological test (IFA) to detect INKV antibodies, and studied protein-specific antibody responses. Antibody prevalence in humans in Finland and Sweden showed that 40-50% of the population had been infected with INKV or a related California serogroup virus. The seroprevalence was higher in older age groups, and in Finland, the prevalence increased northwards. We found that acute-phase sera had a distinct granular fluorescence pattern in IgG IFA, whereas those with pre-existing immunity showed a diffuse pattern. Using recombinant INKV proteins as antigens, the antibody response was showed predominantly to be against the N protein in early infection and also towards the Gc protein in the later stages of infection. We discovered a new California encephalitis virus isolate of the genus Orthobunyavirus in Finland from mosquitoes collected in 2007 and 2008 from the Ilomantsi and Sotkamo municipalities in Eastern Finland. The new isolates were named the Möhkö isolates of Chatanga virus (CHATV) since the genetic and serological findings suggested that the new virus isolates were most closely related to clusters of Russian orthobunyavirus CHATV isolates (99% N protein identity). Using samples from patients with febrile illness or neurological symptoms collected in the summertime between 2001 and 2013, we studied the frequency of acute INKV infections and the clinical picture of the patients. We found the frequency to be under 1%, but interestingly, not only INKV but also CHATV were confirmed to cause human infections. Most patients were not hospitalized, and they had visited a doctor most often once. The patients suffered from fever, headache, vomiting, disorientation, and seizures. INKV infections were more severe in children under 16 years, and CHATV infections were more severe in adults. In conclusion, we found a new mosquito-borne virus from Finland and showed for the first time that this virus was associated with a clinical disease. In addition, we described the clinical picture of INKV infection and showed that the infection is more severe in children if neurologic symptoms appear. These viruses are common in Finland, and their association with clinical disease in the summertime should not be forgotten.
  • Nurmi, Visa (Helsingin yliopisto, 2019)
    In diagnosis of many viral and some other microbial infections, measurement of IgG avidity is a globally emerging approach for identifying primary infections and estimating the time of clinical onset. While many calculation methods have been introduced for conversion of the raw data into a numerical avidity value, little information exists on their comparison and whether the complex and laborious ones show superior performance. We here compare diverse approaches for avidity calculation, and introduce for clinical use a new, highly sensitive and specific one. Enzyme immune assay (EIA) absorbance data from 135 parvovirus B19 IgG-positive sera were analyzed in parallel with the new and two reference methods (Avidity1.2 software representing gold standard methodology; avidity index (AI) widely used due to its simplicity). When sample dilutions were selected on the basis of optimal EIA absorbances, all three approaches performed equally (receiver operating characteristic area-under-curve, AUC, discriminating primary infection from past immunity 0.969-0.971). When the samples were reanalyzed at either lower or higher absorbance levels, avidity status (low, borderline or high) was altered, on the average, in 3.7 % of the samples with the new method, in 5.6 % with Avidity1.2 and in 28 % with AI. The new approach was further validated with extensive serum panels for cytomegalovirus, Toxoplasma gondii, rubella and Epstein–Barr virus (AUC 0.990-1.000). It allows for accurate measurement of antimicrobial IgG avidity even from samples with low IgG level, while the globally popular AI approach provides an alternative for samples with sufficiently high IgG level.
  • Huhtamo, Eili (Helsingin yliopisto, 2010)
    Dengue is a mosquito-borne viral disease caused by the four dengue virus serotypes (DENV-1-4) and is currently considered as the most important arthropod-borne viral disease in the world. Nearly half of the human population lives in risk areas, and 50-100 million infections occur yearly according to World Health Organization. The disease can vary from a mild febrile disease to severe haemorrhagic fever and shock. A secondary infection with heterologous serotype increases the risk for severe disease outcome. During the last three decades the impact of dengue has dramatically increased in the endemic areas including the tropics and subtropics of the world. The current situation with massive epidemics of severe disease forms has been associated with socio-ecological changes that have increased the transmission and enabled the co-circulation of different serotypes. Consequently, an increase of dengue has also been observed in travelers visiting these areas. Currently approximately 30 cases are diagnosed yearly in Finnish travelers. In travelers dengue is rarely a life-threatening disease, however in the current study, a fatality was documented in a young Finnish patient who experienced a prolonged primary dengue infection. To improve particularly early laboratory diagnostics, a novel real-time RT-PCR method was developed for the detection of DENV-1-4 RNA based on TaqMan chemistry. The method was shown to be sensitive and specific for detecting DENV RNA and suitable for diagnostic use. The newly developed real-time RT-PCR was compared to other available early diagnostic methods including IgM and NS1 antigen detection using a panel of selected patient samples. The results suggest that the best diagnostic rates are achieved by a combination of IgM with RNA or NS1 detection. The dengue virus strains studied here included the first DENV strains isolated from serum samples of Finnish travelers collected in 2000-2005. The results of sequence analysis demonstrated that the 11 isolates included all four DENV serotypes and presented a global sample of DENV strains from different geographical areas including Asia, Africa and South America. In the present study sequence analysis was also carried out for a collection of 23 novel DENV-2 isolates from Venezuelan patients collected in 1999-2005. The Venezuelan DENV-2 exclusively represented the American-Asian genotype, suggesting that no foreign DENV-2 lineages have recently been introduced to the country. The results also suggest that the DENV-2 viruses detected earlier from Venezuela have been maintained in the area where they have evolved into several lineages. This is in contrast to the pattern observed in some other dengue endemic areas, where introductions of novel virus types and lineages are frequently detected.
  • Jääskeläinen, Anu (Helsingin yliopisto, 2011)
    Pdf-file, link above
  • Kolehmainen, Pekka (Helsingin yliopisto, 2014)
    Human parechovirus (HPeV) and Ljungan virus (LV) are non-enveloped, single-stranded RNA viruses that form the genus Parechovirus in the family Picornaviridae. The interest in these viruses has notably increased over the past 15 years because of their strengthened associations to human and animal diseases. HPeV types 1 and 3 have been associated with more severe infections in young children, such as infections of the central nervous system (CNS) and sepsis-like disease. Rodent-infecting LV has been suggested to possess zoonotic potential and induce various human diseases. However, the proof for this remains lacking. This study aimed to describe the epidemiological features of HPeVs in Finland and in the Netherlands, to examine the connection between HPeV-induced infection and human diseases and to study the circulation of LV in Finland. The epidemiological analysis of stool samples, collected from 1996 to 2007, revealed that HPeVs are highly common in healthy Finnish children. HPeV was primarily detectable in children under 2 years of age. Altogether, 39% of the study participants tested positive for HPeV at least once during the study period. HPeVs circulated throughout the year, with a distinct seasonal peak in October-November. The results indicated that not only the previously described HPeV1 but also HPeV genotypes 3 and 6 circulate in Finland. Microneutralisation assays, which were set up to detect HPeV1 to 6, the most common genotypes in Europe, provided a deeper understanding of HPeV seroprevalence in the Finnish and Dutch populations. Seropositivity for HPeV1, 2 and HPeV4 to 6 was high and moderate in adults, in contrast to seropositivity for HPeV3, which was extremely low. The serological data demonstrate that HPeV types 1 to 6 might be even more prevalent than previously assumed. All six HPeV types circulate in Finland. In addition to HPeV detection in background populations, we presented the first cases of severe infection in neonates with HPeV4 and, subsequently, the first isolation of this genotype in Finland. Five hospitalised neonates with a sepsis-like disease in the fall of 2012 were positive for HPeV. Four of these children had HPeV4, indicating a potential small epidemic of this genotype, whereas one HPeV remained untyped. In addition, we detected HPeV3 in a neonate with suspected viral sepsis in October 2011 and another untyped HPeV in a child with symptoms corresponding to acute disseminated encephalomyelitis in May 2012. Following these findings, we promoted the addition of HPeV detection to routine diagnostics of young children. To extend the knowledge regarding other parechoviruses in Finland, we studied LV antibody prevalence in both humans and rodents. The seroprevalence detected for LV was 38% in humans and 18% in bank voles (Myodes glareolus). The observation of LV antibodies in humans is relatively high because LV has never been isolated from humans. These results suggest that an LV or LV-like virus, in addition to HPeVs, circulates frequently among human populations in Finland.
  • Tuppurainen, Eeva (Helsingin yliopisto, 2015)
    Lumpy skin disease (LSD) is an economically important pox disease of cattle and Asian water buffalo caused by a lumpy skin disease virus (LSDV), a member of the genus Capripoxvirus. The disease occurs in Africa and the Near East, causing substantial economic losses for the whole cattle industry in affected countries. The disease is characterized by skin nodules, high fever, lymphadenopathy and loss of production of infected animals. Transmission of LSDV is known to occur mechanically by a variety of blood-feeding insects and to a lesser extent through contaminated feed and water, semen or via direct contact. The disease is classified as notifiable by the World Animal Health Organization (OIE). Currently, Finland is free of LSD. The general aim of the study was to investigate the vector capacity of common sub-Saharan tick species, Rhipicephalus appendiculatus, Amblyomma hebraeum and Rhipicephalus decoloratus for LSDV in cattle via mechanical, intra/transstadial or vertical routes. The specific aim was to investigate if mechanical transmission occurs by R. appendiculatus males and transovarial by R. decoloratus females. As many of the infected animals become viraemic without showing skin lesions, it was investigated if feeding on healthy looking skin of viraemic animals was sufficient for successful mechanical transmission. The final objective was to investigate if the virus was able to grow in vitro in Rhipicephalus spp. tick cell lines. In addition, the presence of the virus or viral DNA in ticks collected from naturally infected animals was investigated. Two animal experiments, using naïve cattle and laboratory-reared ticks were conducted at the Department of Veterinary Tropical Diseases, University of Pretoria, South Africa and samples were tested at the Pirbright Institute, United Kingdom. For the first time, transmission of LSDV (or any pox virus) by hard ticks was demonstrated to occur mechanically/intrastadially by R. appendiculatus males and vertically by R. decoloratus females. Feeding directly on skin lesions was not necessary for transmission of the virus between infected and naïve cattle. No evidence of viral replication in Rhipicephalus tick cell lines was obtained. The presence of the viral DNA was detected in Rhipicaphalus, Amblyomma and Hyalomma ticks collected during natural LSDV outbreaks in South Africa and Egypt. In 2013 - 2015 LSDV is spreading in the Near East at a scale never seen before, posing a threat to the European Union, Caucasus region, Afghanistan and Pakistan. In order to control and eradicate the disease, it is fundamental to understand the role of different arthropod vectors and their importance in the field. The presence of infected tick eggs or different instars in the environment underlines the importance of effective prophylactic tools and sufficient vaccination coverage. In addition, this study contributes to the recommendations set for the international trade of live cattle from affected countries.
  • Katz, Anna (Helsingin yliopisto, 2012)
    The Uukuniemi virus (UUKV) is a member of the Bunyaviridae family (genus Phlebovirus). The virus was isolated from Ixodes ricinus ticks from Uukuniemi, Finland in 1959 and was found to be non-pathogenic for humans. UUKV has served for more than four decades as an excellent model to study the molecular and cellular biology of the serious human pathogens that reside within this group. UUKV has a segmented, single-stranded RNA genome of negative polarity. The three RNA segments (S, M, and L) encode four structural proteins: a nucleocapsid (N) protein, two glycoproteins (Gn and Gc), and an RNA-dependent RNA polymerase (L protein). In addition, a non-structural protein (NSs) is encoded from the S segment using an ambisense coding strategy. At the termini of the RNA segments, there are non-coding regions, which contain regulatory elements for viral transcription and replication. The very terminal 5' and 3' ends within all non-coding regions are complementary to each other, and highly conserved within the genus. In order to function as templates for transcription and replication, all three RNA segments must be encapsidated by the N protein. The N protein forms oligomers, in which N protein molecules are bound to each other; this oligomer associates with RNA. This study focused on analyzing the function of the non-coding regions in the termini of the RNA segments, and on locating amino acid residues or domains of the UUKV N protein, which could potentially be involved in the oligomerization or RNA-binding. The function of the non-coding regions was studied using a minigenome system developed for UUKV, where the viral protein coding sequence is replaced by sequences encoding a reporter protein. The cells are transfected with the minigenomes and helper plasmids, and after replication and transcription of the minigenomes, the reporter protein expression can be measured. The non-coding regions of all three RNA segments were analyzed and promoter strengths and packaging efficiencies were compared. The results showed that the non-coding regions in all three RNA segments contain all the necessary signals for initiation of transcription and replication and encapsidation and packaging of the RNA segments. The strongest promoter strength was observed in M segment, followed by L and S segments. The role of the intergenic region, which is located between the N and NSs genes in the UUKV S segment was also analyzed and was found to regulate termination of transcription. To study the oligomerization and RNA-binding of UUKV N protein, a set of N protein mutants were generated based on 2D and 3D predictions of the N protein. The functionality of these mutants was analyzed using mammalian two-hybrid-, minigenome-, and virus-like particle-assays, which showed that both the N- and C-termini of the N protein are needed for the oligomerization. A specific structure in the N-terminal region plays an important role in the N-N interactions. Some putative RNA-binding residues were found, which severely affected the N protein functionality in all three assays. These residues were located within the proposed RNA-binding cavity in the predicted UUKV N protein models. These results are in agreement with observations with other bunyaviruses, and could help to better understand the molecular biology of bunyaviruses. Moreover, understanding the details of the oligomerization and RNA-binding of the N protein could help in design of potential antivirals for the pathogenic phleboviruses.
  • Kivi, Niina (Helsingin yliopisto, 2011)
    Human papillomaviruses (HPVs) are the causal agents of cervical cancer, which is the second most common cancer among women worldwide. Cellular transformation and carcinogenesis depend on the activities of viral E5, E6 and E7 proteins. Alterations in cell-cell contacts and in communication between epithelial cells take place during cervical carcinogenesis, leading to changes in cell morphology, increased cell motility and finally invasion. The aim of this thesis was to study genome-wide effects of the HPV type 16 (HPV-16) E5 protein on the expression of host cell messenger RNAs (mRNAs) and microRNAs by applying microarray technology. The results showed that the HPV-16 E5 protein alters several cellular pathways involved in cellular adhesion, motility and proliferation as well as in the extracellular matrix. The E5 protein was observed to enhance wound healing of epithelial cell monolayers by increasing cell motility in vivo. HPV-16 E5-induced alterations in the expression of cellular microRNAs and their target genes seem to favour increased proliferation and tumorigenesis. E5 was also shown to affect the expression of adherens junction proteins in HaCaT epithelial keratinocytes. In addition, a study of a membrane cytoskeletal cross-linker protein, ezrin, revealed that when activated, it localizes to adherens junctions. The results suggest that ezrin distribution to forming adherens junctions is due to Rac1 activity in epithelial cells. These studies reveal for the first time the holistic effects of HPV-16 E5 protein in promoting precancerous events in epithelial cells. The results contribute to identifyinging novel markers for cervical precancerous stages and to predicting disease behaviour.
  • Hepojoki, Jussi (Helsingin yliopisto, 2011)
    Hantaviruses are one of the five genera of the vector-borne virus family Bunyaviridae. While other members of the family are transmitted via arthropods, hantaviruses are carried and transmitted by rodents and insectivores. Occasional transmission to humans occurs via inhalation of aerosolized rodent excreta. When transmitted to man hantaviruses cause hemorrhagic fever with renal syndrome (HFRS, in Eurasia, mortality ~10%) and hantavirus cardiopulmonary syndrome (HCPS, in the Americas, mortality ~40%). The single-stranded, negative-sense RNA genome of hantaviruses is in segments S, M and L that respectively encode for nucleocapsid (N), glycoproteins Gn and Gc, and RNA-dependent RNA-polymerase (RdRp or L protein). The genome segments, encapsidated by N protein to form ribonucleoprotein (RNP), are enclosed inside a lipid envelope decorated by spikes formed of Gn and Gc. The focus of this study was to understand the mechanisms and interactions through which the virion is formed and maintained. We observed that when extracted from virions both Gn and Gc favor homo- over hetero-oligomerization. The minimal glycoprotein complexes extracted from virion by detergent were observed, by using ultracentrifugation and gel filtration, to be tetrameric Gn and homodimeric Gc. These results led us to suggest a model where tetrameric Gn complexes are interconnected through homodimeric Gc units to form the grid-like surface architecture described for hantaviruses. This model was found to correlate with the three-dimensional (3D) reconstruction of virion surface created using cryo-electron tomography (cryo-ET). The 3D-density map showed the spike complex formed of Gn and Gc to be 10 nm high and to display a four-fold symmetry with dimensions of 15 nm times 15 nm. This unique square-shaped complex on a roughly round virion creates a hitch for the assembly, since a sphere cannot be broken into rectangles. Thus additional interactions are likely required for the virion assembly. In cryo-ET we observed that the RNP makes occasional contacts to the viral membrane, suggesting an interaction between the spike and RNP. We were able to demonstrate this interaction using various techniques, and showed that both Gn and Gc contribute to the interaction. This led us to suggest that in addition to the interactions between Gn and Gc, also the interaction between spike and RNP is required for assembly. We found galectin-3 binding protein (referred to as 90K) to co-purify with the virions and showed an interaction between 90K and the virion. Analysis of plasma samples taken from patients hospitalized for Puumala virus infection showed increased concentrations of 90K in the acute phase and the increased 90K level was found to correlate with several parameters that reflect the severity of acute HFRS. The results of these studies confirmed, but also challenged some of the dogmas on the structure and assembly of hantaviruses. We confirmed that Gn and RNP do interact, as long assumed. On the other hand we demonstrated that the glycoproteins Gn and Gc exist as homo-oligomers or appear in large hetero-oligomeric complexes, rather than form primarily heterodimers as was previously assumed. This work provided new insight into the structure and assembly of hantaviruses.
  • Strandin, Tomas (Helsingin yliopisto, 2011)
    Hantaviruses (family Bunyaviridae, genus Hantavirus) are enveloped viruses incorporating a segmented, negative-sense RNA genome. Each hantavirus is carried by its specific host, either a rodent or an insectivore (shrew), in which the infection is asymptomatic and persistent. In humans, hantaviruses cause Hemorrhagic fever with renal syndrome (HFRS) in Eurasia and Hantavirus cardiopulmonary syndrome (HCPS) in the Americas. In Finland, Puumala virus (genus Hantavirus) is the causative agent of NE, a mild form of HFRS. The HFRS-type diseases are often associated with renal failure and proteinuria that might be mechanistically explained by infected kidney tubular cell degeneration in patients. Previously, it has been shown that non-pathogenic hantavirus, Tula virus (TULV), could cause programmed cell death, apoptosis, in cell cultures. This suggested that the infected kidney tubular degeneration could be caused directly by virus replication. In the first paper of this thesis the molecular mechanisms involved in TULV-induced apoptosis was further elucidated. A virus replication-dependent down-regulation of ERK1/2, concomitantly with the induced apoptosis, was identified. In addition, this phenomenon was not restricted to TULV or to non-pathogenic hantaviruses in general since also a pathogenic hantavirus, Seoul virus, could inhibit ERK1/2 activity. Hantaviruses consist of membrane-spanning glycoproteins Gn and Gc, RNA-dependent RNA polymerase (L protein) and nucleocapsid protein N, which encapsidates the viral genome, and thus forms the ribonucleoprotein (RNP). Interaction between the cytoplasmic tails of viral glycoproteins and RNP is assumed to be the only means how viral genetic material is incorporated into infectious virions. In the second paper of this thesis, it was shown by immunoprecipitation that viral glycoproteins and RNP interact in the purified virions. It was further shown that peptides derived from the cytoplasmic tails (CTs) of both Gn and Gc could bind RNP and recombinant N protein. In the fourth paper the cytoplamic tail of Gn but not Gc was shown to interact with genomic RNA. This interaction was probably rather unspecific since binding of Gn-CT with unrelated RNA and even single-stranded DNA were also observed. However, since the RNP consists of both N protein and N protein-encapsidated genomic RNA, it is possible that the viral genome plays a role in packaging of RNPs into virions. On the other hand, the nucleic acid-binding activity of Gn may have importance in the synthesis of viral RNA. Binding sites of Gn-CT with N protein or nucleic acids were also determined by peptide arrays, and they were largely found to overlap. The Gn-CT of hantaviruses contain a conserved zinc finger (ZF) domain with an unknown function. Some viruses need ZFs in entry or post-entry steps of the viral life cycle. Cysteine residues are required for the folding of ZFs by coordinating zinc-ions, and alkylation of these residues can affect virus infectivity. In the third paper, it was shown that purified hantavirions could be inactivated by treatment with cysteine-alkylating reagents, especially N-ethyl maleimide. However, the effect could not be pin-pointed to the ZF of Gn-CT since also other viral proteins reacted with maleimides, and it was, therefore, impossible to exclude the possibility that other cysteines besides those that were essential in the formation of ZF are required for hantavirus infectivity.
  • Virtanen, Oskari (Helsingin yliopisto, 2009)
    Human herpesvirus 6 (HHV-6) was identified from patients with HIV and lymphoproliferative diseases in 1986. It is a β-herpesvirus and is divided into two subgroups, variants A and B. HHV-6 variant B is the cause of exanthema subitum, while variant A has not yet definitely proven to cause any disease. HHV-6, especially variant A, is a highly neurotropic virus and has been associated with many diseases of the central nervous system (CNS) such as encephalitis and multiple sclerosis (MS). The present studies were aimed to elucidate the role of HHV-6 and its two variants in neurological infections. Special attention was given to study the possible role of HHV-6 in the pathogenesis of MS. We studied the expression of HHV-6 antigens using immunohistochemistry in brain autopsy samples from patients with MS and controls. HHV-6 antigen was identified in 70% of MS specimens whereas 30% of control specimens expressed HHV-6 antigen. Serum and cerebrospinal fluid (CSF) samples were collected from patients with MS and patients with other neurological diseases (OND) from patients visiting Helsinki University Central Hospital Neurological Outpatient Clinic during the years 2003 and 2004. In addition, we studied 53 children with suspected encephalitis. We developed an immunofluorescence IgG-avidity assay for the detection of primary HHV-6A and HHV-6B infection. For HHV-6B antibodies, no differences were observed between patients with MS and OND. For HHV-6A both seroprevalence and mean titers were significantly higher in MS compared to OND. HHV-6A low-avidity IgG antibodies, suggestive of primary infection, were found in serum of two, three and one patient with definite MS, possible MS and OND, respectively. From pediatric patients with suspected encephalitis, six serum samples (11.3%) contained low-avidity antibodies, indicating a temporal association between HHV-6A infection and onset of encephalitis. Three out of 26 patients with CDMS and four out of 19 patients with CPMS had HHV-6 antibodies in their CSF compared to none of the patients with OND (p=0.06 and p=0.01, respectively). Two patients with CDMS and three patients with CPMS appeared to have specific intrathecal synthesis of HHV-6A antibodies. In addition, oligoclonal bands (OCB) were observed in the CSF of five out of nine MS patients tested, and in two the OCBs reacted specifically with HHV-6 antigen, which is a novel finding. These results indicate HHV-6 specific antibody production in the CNS and suggest that there is a subset of MS patients with an active or chronic HHV-6A infection in the CNS that might be involved in the pathogenesis of MS. Our studies suggest that HHV-6 is an important causative or associated virus in some neurological infections, such as encephalitis and it might contribute to the development of MS, at least in some cases. In conclusion, HHV-6 is a neurotropic virus that should be taken into consideration when studying acute and chronic CNS diseases of unknown origin.
  • Hokynar, Kati (Helsingin yliopisto, 2007)
    Human parvovirus B19 is a minute ssDNA virus causing a wide variety of diseases, including erythema infectiosum, arthropathy, anemias, and fetal death. After primary infection, genomic DNA of B19 has been shown to persist in solid tissues of not only symptomatic but also of constitutionally healthy, immunocompetent individuals. In this thesis, the viral DNA was shown to persist as an apparently intact molecule of full length, and without persistence-specific mutations. Thus, although the mere presence of B19 DNA in tissue can not be used as a diagnostic criterion, a possible role in the pathogenesis of diseases e.g. through mRNA or protein production can not be excluded. The molecular mechanism, the host-cell type and the possible clinical significance of B19 DNA tissue persistence are yet to be elucidated. In the beginning of this work, the B19 genomic sequence was considered highly conserved. However, new variants were found: V9 was detected in 1998 in France, in serum of a child with aplastic crisis. This variant differed from the prototypic B19 sequences by ~10 %. In 2002 we found, persisting in skin of constitutionally healthy humans, DNA of another novel B19 variant, LaLi. Genetically this variant differed from both the prototypic sequences and the variant V9 also by ~10%. Simultaneously, B19 isolates with DNA sequences similar to LaLi were introduced by two other groups, in the USA and France. Based on phylogeny, a classification scheme based on three genotypes (B19 types 1-3) was proposed. Although the B19 virus is mainly transmitted via the respiratory route, blood and plasma-derived products contaminated with high levels of B19 DNA have also been shown to be infectious. The European Pharmacopoeia stipulates that, in Europe, from the beginning of 2004, plasma pools for manufacture must contain less than 104 IU/ml of B19 DNA. Quantitative PCR screening is therefore a prerequisite for restriction of the B19 DNA load and obtaining of safe plasma products. Due to the DNA sequence variation among the three B19 genotypes, however, B19 PCR methods might fail to detect the new variants. We therefore examined the suitability of the two commercially available quantitative B19 PCR tests, LightCycler-Parvovirus B19 quantification kit (Roche Diagnostics) and RealArt Parvo B19 LC PCR (Artus), for detection, quantification and differentiation of the three B19 types known, including B19 types 2 and 3. The former method was highly sensitive for detection of the B19 prototype but was not suitable for detection of types 2 and 3. The latter method detected and differentiated all three B19 virus types. However, one of the two type-3 strains was detected at a lower sensitivity. Then, we assessed the prevalence of the three B19 virus types among Finnish blood donors, by screening pooled plasma samples derived from >140 000 blood-donor units: none of the pools contained detectable levels of B19 virus types 2 or 3. According to the results of other groups, B19 type 2 was absent also among Danish blood-donors, and extremely rare among symptomatic European patients. B19 type 3 has been encountered endemically in Ghana and (apparently) in Brazil, and sporadical cases have been detected in France and the UK. We next examined the biological characteristics of these virus types. The p6 promoter regions of virus types 1-3 were cloned in front of a reporter gene, the constructs were transfected into different cell lines, and the promoter activities were measured. As a result, we found that the activities of the three p6 promoters, although differing in sequence by >20%, were of equal strength, and most active in B19-permissive cells. Furthermore, the infectivity of the three B19 types was examined in two B19-permissive cell lines. RT-PCR revealed synthesis of spliced B19 mRNAs, and immunofluorescence verified the production of NS1 and VP proteins in the infected cells. These experiments suggested similar host-cell tropism and showed that the three virus types are strains of the same species, i.e. human parvovirus B19. Last but not least, the sera from subjects infected in the past either with B19 type 1 or type 2 (as evidenced by tissue persistence of the respective DNAs), revealed in VP1/2- and VP2-EIAs a 100 % cross-reactivity between virus types 1 and 2. These results, together with similar studies by others, indicate that the three B19 genotypes constitute a single serotype.
  • Kakkola, Laura (Helsingin yliopisto, 2008)
    Torque teno virus (TTV) was discovered in 1997 in the serum of a Japanese patient who had a post-transfusion hepatitis of unknown etiology. It is a small virus containing a circular single-stranded DNA genome which is unique among human viruses. Within a few years after its discovery, the TTVs were noted to form a large family of viruses with numerous genotypes. TTV is highly prevalent among the general population throughout the world, and persistent infections and co-infections with several genotypes occur frequently. However, the pathogenicity and the mechanism for the sustained occurrence of the virus in blood are at present unclear. To determine the prevalence of TTV in Finland, we set up PCR methods and examined the sera of asymptomatic subjects for the presence of TTV DNA and for genotype-6 DNA. TTV was found to be highly prevalent also in Finland; 85% of adults harbored TTV in their blood, and 4% were infected with genotype-6. In addition, TTV DNA was detected in a number of different tissues, with no tissue-type or symptom specificity. Most cell-biological events during TTV infections are at the moment unknown. Replicating TTV DNA has, however, been detected in liver and the hematopoietic compartment, and three mRNAs are known to be generated. To characterize TTV cell biology in more detail, we cloned in full length the genome of TTV genotype 6. We showed that in human kidney-derived cells TTV produces altogether six proteins with distinct subcellular localizations. TTV mRNA transcription was detected in all cell lines transfected with the full-length clone, and TTV DNA replicated in several of them, including those of erythroid, kidney, and hepatic origin. Furthermore, the viral DNA replication was shown to utilize the cellular DNA polymerases. Diagnoses of TTV infections have been based almost solely on PCR, whereas serological tests, measuring antibody responses, would give more information on many aspects of these infections. To investigate the TTV immunology in more detail, we produced all six TTV proteins for use as antigens in serological tests. We detected in human sera IgM and IgG antibodies to occur simultaneously with TTV DNA, and observed appearance of TTV DNA regardless of pre-existing antibodies, and disappearance of TTV DNA after antibody appearance. The genotype-6 nucleotide sequence remained stable for years within the infected subjects, suggesting that some mechanism other than mutations is used by this minute virus to evade our immune system and to establish chronic infections in immunocompetent subjects.
  • Jaatinen, Silja (Silja Jaatinen, 2009)
    In this thesis three icosahedral lipid-containing double-stranded (ds) deoxyribonucleic acid (DNA) bacteriophages have been studied: PRD1, Bam35 and P23-77. The work focuses on the entry, exit and structure of the viruses. PRD1 is the type member of the Tectiviridae family, infecting a variety of Gram-negative bacteria. The PRD1 receptor binding complex, consisting of the penton protein P31, the spike protein P5 and the receptor binding protein P2 recognizes a specific receptor on the host surface. In this study we found that the transmembrane protein P16 has an important stabilization function as the fourth member of the receptor binding complex and protein P16 may have a role in the formation of a tubular membrane structure, which is needed in the ejection of the genome into the cell. Phage Bam35 (Tectiviridae), which infects Gram-positive hosts, has been earlier found to resemble PRD1 in morphology and genome organization The uncharacterized early and late events in the Bam35 life cycle were studied by electrochemical methods. Physiological changes in the beginning of the infection were found to be similar in both lysogenic and nonlysogenic cell lines, Bam35 inducing a temporal decrease of membrane voltage and K+ efflux. At the end of the infection cycle physiological changes were observed only in the nonlysogenic cell line. The strong K+ efflux 40 min after infection and the induced premature cell lysis propose that Bam35 has a similar holin-endolysin lysis system to that of PRD1. Thermophilic icosahedral dsDNA Thermus phages P23-65H, P23-72 and P23-77 have been proposed to belong to the Tectiviridae family. In this study these phages were compared to each other. Analysis of structural protein patterns and stability revealed these phages to be very similar but not identical. The most stable of the studied viruses, P23-77, was further analyzed in more detail. Cryo-electron microscopy and three-dimensional image reconstruction was used to determine the structure of virus to 14 Å resolution. Results of thin layer chromatography for neutral lipids together with analysis of the three dimensional reconstruction of P23-77 virus particle revealed the presence of an internal lipid membrane. The overall capsid architecture of P23-77 is similar to PRD1 and Bam35, but most closely it resembles the structure of the capsid of archaeal virus SH1. This complicates the classification of dsDNA, internal lipid-containing icosahedral viruses.
  • Razzauti Sanfeliu, Maria (Helsingin yliopisto, 2012)
    Puumala hantavirus (PUUV) is a zoonotic virus that in humans causes nephropathia epidemica (NE) in humans, a mild form of haemorrhagic fever with renal syndrome. An average of 10 000 cases are reported annually in Europe, many of which occur in Fennoscandia. The incidence of NE is connected to the distribution and population density of the the bank vole (Myodes glareolus), the main virus host. In Fennoscandia, high incidences of NE occur at 3-4 year intervals due to the characteristic population cycles of this woodland rodent. This study aimed to gain a better understanding of PUUV microevolution by examining genetic features of the virus in several bank vole populations of Finland and Latvia. Genetic variation in PUUV circulating in a bank vole population at Konnevesi in Central Finland was examined and monitored over five-years throughout a complete bank vole cycle, including two peak-phases in 2005 and 2008 and two population declines in 2006 and 2009 (i.e., viral bottlenecks). Altogether, 1369 bank voles were captured and 26.3% were detected PUUV-infected. Partial sequences of the three viral genome segments (Small, Medium and Large) were inspected from 365 PUUV genomes. Genetic diversity was 6.2% for the S segment, 4.8% for the M segment, and a surprisingly high 10.1% for the L segment. Each genome segment had accumulated mutations as a separate gene pool. The majority of nucleotide substitutions were synonymous and most of the deduced amino acid substitutions were conservative, suggesting a strong stabilizing selection operating at the protein level. Genetic markers found along the genome segments allowed for the recognition of two genogroups of PUUV co-circulating in the host population. Even though, one of the genogroups presented higher genetic diversity, no signs of completion were observed between them. Nearly 80% the variants exhibited a transient existence, and frequently occurring variants were integrated by most abundant segment genotypes suggesting a viral mutational robustness. A substantial portion (19.1%) of genomes appeared to be reassorted, with S and M typically being exchanged. Reassorted variants did not outcompete parental variants and were commonly transient. Reassortment was seasonal, occurring more frequently in autumn when recent infection risk increases. An imperceptible intra-genogroup reassortment could contribute to the steady state of the viral population, counteracting the effects of Muller s ratchet. Co-circulation and interaction of two distinct PUUV lineages (Finnish and North-Scandinavian) was monitored in a bank vole population at Pallasjärvi in Northern Finland. To date, seven genetic lineages have been detected, all of which exhibit geographic structure within the host distribution. Here, we present new evidence of two lineages circulating in the same bank vole phylogroup (Ural clade). Genetic diversity within each PUUV lineage was modest (up to 1.7%) and most substitutions were synonymous. However, genetic differences between the two lineages were as high as 18.9%. Phylogenetic analyses revealed that these distinct lineages naturally reassort with a frequency comparable to that genogroups circulating at Konnevesi, i.e., 32%. In contrast to Konnevesi, only M segment was exchanged between PUUV lineages at Pallasjärvi. Two distinct PUUV lineages were also found to co-circulate in Latvia. One (Russian) has been previously described and the other awaits formal description. The novel Latvian lineage is considerably divergent from other PUUV lineages and several amino acid markers made it easily distinguishable. Phylogenetic analysis suggested an independent evolutionary history for the segments of Latvian lineage. Similar to Pallasjärvi, both Russian and Latvian lineages were found in a single bank vole phylogroup (Carpathian clade), confirming earlier observations that PUUV lineages are not limited to a single host phylogroup.
  • Savolainen-Kopra, Carita (Helsingin yliopisto, 2006)
    The first part of this work investigates the molecular epidemiology of a human enterovirus (HEV), echovirus 30 (E-30). This project is part of a series of studies performed in our research team analyzing the molecular epidemiology of HEV-B viruses. A total of 129 virus strains had been isolated in different parts of Europe. The sequence analysis was performed in three different genomic regions: 420 nucleotides (nt) in the VP4/VP2 capsid protein coding region, the entire VP1 capsid protein coding gene of 876 nt, and 150 nt in the VP1/2A junction region. The analysis revealed a succession of dominant sublineages within a major genotype. The temporally earlier genotypes had been replaced by a genetically homogenous lineage that has been circulating in Europe since the late 1970s. The same genotype was found by other research groups in North America and Australia. Globally, other cocirculating genetic lineages also exist. The prevalence of a dominant genotype makes E-30 different from other previously studied HEVs, such as polioviruses and coxsackieviruses B4 and B5, for which several coexisting genetic lineages have been reported. The second part of this work deals with molecular epidemiology of human rhinoviruses (HRVs). A total of 61 field isolates were studied in the 420-nt stretch in the capsid coding region of VP4/VP2. The isolates were collected from children under two years of age in Tampere, Finland. Sequences from the clinical isolates clustered in the two previously known phylogenetic clades. Seasonal clustering was found. Also, several distinct serotype-like clusters were found to co-circulate during the same epidemic season. Reappearance of a cluster after disappearing for a season was observed. The molecular epidemiology of the analyzed strains turned out to be complex, and we decided to continue our studies of HRV. Only five previously published complete genome sequences of HRV prototype strains were available for analysis. Therefore, all designated HRV prototype strains (n=102) were sequenced in the VP4/VP2 region, and the possibility of genetic typing of HRV was evaluated. Seventy-six of the 102 prototype strains clustered in HRV genetic group A (HRV-A) and 25 in group B (HRV-B). Serotype 87 clustered separately from other HRVs with HEV species D. The field strains of HRV represented as many as 19 different genotypes, as judged with an approximate demarcation of a 20% nt difference in the VP4/VP2 region. The interserotypic differences of HRV were generally similar to those reported between different HEV serotypes (i.e. about 20%), but smaller differences, less than 10%, were also observed. Because some HRV serotypes are genetically so closely related, we suggest that the genetic typing be performed using the criterion "the closest prototype strain". This study is the first systematic genetic characterization of all known HRV prototype strains, providing a further taxonomic proposal for classification of HRV. We proposed to divide the genus Human rhinoviruses into HRV-A and HRV-B. The final part of the work comprises a phylogenetic analysis of a subset (48) of HRV prototype strains and field isolates (12) in the nonstructural part of the genome coding for the RNA-dependent RNA polymerase (3D). The proposed division of the HRV strains in the species HRV-A and HRV-B was also supported by 3D region. HRV-B clustered closer to HEV species B, C, and also to polioviruses than to HRV-A. Intraspecies variation within both HRV-A and HRV-B was greater in the 3D coding region than in the VP4/VP2 coding region, in contrast to HEV. Moreover, the diversity of HRV in 3D exceeded that of HEV. One group of HRV-A, designated HRV-A', formed a separate cluster outside other HRV-A in the 3D region. It formed a cluster also in the capsid region, but located within HRV-A. This may reflect a different evolutionary history of distinct genomic regions among HRV-A. Furthermore, the tree topology within HRV-A in the 3D region differed from that in the VP4/VP2, suggesting possible recombination events in the evolution of the strains. No conflicting phylogenies were observed in any of the 12 field isolates. Possible recombination was further studied using the Similarity and Bootscanning analyses of the complete genome sequences of HRV available in public databases. Evidence for recombination among HRV-A was found, as HRV2 and HRV39 showed higher similarity in the nonstructural part of the genome. Whether HRV2 and HRV39 strains - and perhaps also some other HRV-A strains not yet completely sequenced - are recombinants remains to be determined.
  • Sarin, Peter (2010)
    Double-stranded RNA (dsRNA) viruses encode only a single protein species that contains RNA-dependent RNA polymerase (RdRP) motifs. This protein is a central component in the life cycle of a dsRNA virus, carrying out both RNA transcription and replication. The architecture of viral RdRPs resembles that of a 'cupped right hand' with fingers, palm and thumb domains. Those applying de novo initiation have additional structural features, including a flexible C-terminal domain that constitutes the priming platform. Moreover, viral RdRPs must be able to interact with the incoming 3'-terminus of the template and position it so that a productive binary complex is formed. Bacteriophage phi6 of the Cystoviridae family is to date one of the best studied dsRNA viruses. The purified recombinant phi6 RdRP is highly active in vitro and possesses both RNA replication and transcription activities. The extensive biochemical observations and the atomic level crystal structure of the phi6 RdRP provides an excellent platform for in-depth studies of RNA replication in vitro. In this thesis, targeted structure-based mutagenesis, enzymatic assays and molecular mapping of phi6 RdRP and its RNA were used to elucidate the formation of productive RNA-polymerase binary complexes. The positively charged rim of the template tunnel was shown to have a significant role in the engagement of highly structured ssRNA molecules, whereas specific interactions further down in the template tunnel promote ssRNA entry to the catalytic site. This work demonstrated that by aiding the formation of a stable binary complex with optimized RNA templates, the overall polymerization activity of the phi6 RdRP can be greatly enhanced. Furthermore, proteolyzed phi6 RdRPs that possess a nick in the polypeptide chain at the hinge region, which is part of the extended loop, were better suited for catalysis at higher temperatures whilst favouring back-primed initiation. The clipped C-terminus remains associated with the main body of the polymerase and the hinge region, although structurally disordered, is involved in the control of C-terminal domain displacement. The accumulated knowhow on bacteriophage phi6 was utilized in the development of two technologies for the production of dsRNA: (i) an in vitro system that combines the T7 RNA polymerase and the phi6 RdRP to generate dsRNA molecules of practically unlimited length, and (ii) an in vivo RNA replication system based on restricted infection with phi6 polymerase complexes in bacterial cells to produce virtually unlimited amounts of dsRNA. The pools of small interfering RNAs derived from dsRNA produced by these systems were validated and shown to efficiently decrease the expression of both exogenous and endogenous targets.
  • Jokela, Pia (Helsingin yliopisto, 2012)
    The family Picornaviridae includes many human pathogens. Human enteroviruses (HEVs) exhibit a variety of clinical manifestations ranging from poliomyelitis and encephalomyelitis to respiratory infections and rashes. Human rhinoviruses (HRVs) are the major causes of the common cold. Human parechoviruses (HPeVs) and Aichi virus (AV) are mostly detected in cases of gastroenteritis, and hepatitis A virus (HAV) causes hepatitis with favourable prognosis. In addition to HEVs and HRVs, a large number of viruses are recognized as respiratory pathogens. The conventional respiratory pathogens include influenza A and B viruses, human respiratory syncytial virus (RSV), adenoviruses (AdVs), parainfluenza viruses (PIVs) and the human coronaviruses (hCoVs) OC43 and 229E. Moreover, several new respiratory pathogens, such as human metapneumovirus (hMPV), severe acute respiratory syndrome coronavirus (SARS-CoV), and the hCoVs HKU1 and NL63 have been found during the 2000s. Human bocavirus (hBoV) is also increasingly being recognized as a true pathogen of humans. Since many clinical illnesses may be caused by several different viruses, multiplex assays for simultaneous detection of several viruses are increasingly being applied. Real-time multiplex polymerase chain reaction (PCR) assays for detection of viral nucleic acids offer remarkable benefits, such as short turnaround time and the non-necessity for handling amplified products. Since multiplexing, utilizing real-time PCR, is limited by reduction in amplification efficiency due to multiple primer and probe sets, separate amplification and hybridization reactions have re-emerged in attempts to develop tests with broad diagnostic range. With this approach microarrays, which have the potential for resolving complex mixtures of amplification products, may be applied. In this study, a multiplex reverse transcription-PCR (RT-PCR) and liquid hybridization assay for sensitive detection of HEV, HRV, HPeV and AV were developed and a single RT-PCR and liquid hybridization assay for detection of HAV was optimized. In analysis of clinical samples, the results obtained by the multiplex assay were consistent with those obtained by routine diagnostic assays. When 68 stool samples were analysed for the presence of HPeV and AV, one sample positive for HPeV was detected. This finding is in line with the current knowledge of neither of these viruses being very common enteric pathogens. More rapid detection of HEV and HRV in respiratory samples was achieved when a real-time duplex RT-PCR assay for detection of these viruses was developed. The same approach was used to develop another assay for more sensitive detection of RSV than with the direct fluorescent assay (DFA) and simultaneous identification of hMPV. Both multiplex real-time RT-PCR assays provided reliable and sensitive detection of their targets, except for detection of HRV, since doubts were raised on the ability of the assay to detect all rhinoviruses. Moreover, two commercial hMPV antibodies were found applicable for detection of the virus in respiratory samples by DFA. Results from analysis of respiratory samples using the duplex real-time RT-PCR assays were compared with those obtained with DFA and the Respiratory Viral Panel (RVP) Fast test, a bead-based suspension microarray test evaluated for routine diagnosis. The RVP Fast assay and PCR showed similar detection rates, except for HEV/HRV, for which a higher detection rate by RVP was observed. All PCR-based assays presented more findings of their target viruses than DFA. The broad detection range of the RVP Fast assay resulted in a nearly threefold overall detection rate, compared with that by DFA. Moreover, analysis of clinical samples resulted in a notable prevalence of hMPV and non-SARS-hCoVs, which emphasizes the role of these viruses as respiratory pathogens. Although the RVP Fast assay demonstrated adequate overall performance, doubts were raised on the ability of the test to detect the H1N1 2009 influenza A virus and all AdV serotypes. Evaluation of the RVP Fast assay demonstrated the remarkable increase in overall viral detection rate that results from adapting a PCR-based multiplex assay to virus diagnostics. The sensitive detection of all the viruses of clinical relevance facilitates efficient infection control measures and appropriate patient management and enables systematic studies on the clinical importance of coinfections. Moreover, collection of data on occurrence of all the viruses of clinical relevance will enable a better understanding of the seasonality, geographical distribution and risk groups of the viral pathogens.
  • Väisänen, Elina (Helsingin yliopisto, 2020)
    In the beginning of the 21st century, the development of next generation sequencing (NGS) methods revolutionized the discovery of novel viruses and laid the foundations of a new era in virology. With NGS, the amount and quality of sequence data increased tremendously, and the decreasing price made large-scale screening possible. As a result, hundreds of novel viruses have been identified, and especially the number of novel small DNA viruses, including parvoviruses, has grown substantially. This thesis consists of studies of three novel parvoviruses, bufavirus (BuV), tusavirus (TuV), and cutavirus (CuV). All three viruses were originally identified by NGS from the diarrheal feces of children between 2012 and 2016, and they were the first viruses in the Protoparvovirus genus putatively infecting humans. Many of the animal viruses in this genus are known pathogens: for example, canine parvovirus (CPV) can cause severe gastroenteritis with high mortality among puppies. Currently, there are three genotypes of BuV and one genotype each of CuV and TuV. BuVs have been detected mainly in fecal samples, whereas CuV DNA has been detected in skin biopsies of cutaneous T-cell lymphoma (CTCL) and melanoma patients as well. Any findings of TuV have been rare. Here, we developed diagnostic methods for detecting viral DNA and IgG antibodies against BuV1-3, TuV and CuV, and analyzed 4300 samples from 11 cohorts on four continents to determine the epidemiology and potential clinical impact of these viruses. The BuV DNA was detected in diarrheal fecal samples from both adults and children in Finland, although with low prevalence. In addition, one nasal swab harbored BuV DNA. These studies were among the first to show BuV circulation in Europe and among adults, and further, the nasal swab is still the only non-fecal human sample containing BuV DNA. Although we detected BuV exclusively in diarrheic feces, our results did not strongly support a major role of BuV in gastroenteritis. One of the main findings in this thesis was the remarkable difference observed in the BuV seroprevalence between different populations: in the Middle East and Africa, the BuV IgG antibodies were detected in 56-85% of the adult population whereas in Finland and in the USA the seroprevalence was very low, <4%. This indicated that Iraq, Iran and Kenya are endemic areas for BuV. In addition to the seroprevalence difference, the predominant BuV type varied: it was BuV1 in the Middle East and BuV3 in Kenya. In Kenya, an age-dependent increase in BuV seroprevalence in children was apparent, and it seems that BuVs are not infecting young children in particular. The next important step in BuV studies will be identifying patients with acute primary infection to elucidate the symptoms and clinical picture of the infection. Our data on the geographical distribution of BuVs will help defining suitable locations for such studies. In contrast to that of BuV, the CuV seroprevalence was low in all populations analyzed. Therefore, it is interesting that CuV DNA was detected significantly more often in the lesional skin biopsies from CTCL patients in Finland than in those of transplant patients or healthy adults. This indicates that there is an association between the CuV DNA presence and CTCL, however, it is not known whether this association is causal, accidental or something else. The analysis of additional skin biopsies from the CuV DNA-positive patients revealed CuV DNA in every available skin sample, including both healthy and malignant tissue. Furthermore, lymph nodes harbored CuV DNA, while the prostate samples were CuV negative. Serological analysis of archived serum samples showed that the patients with CuV DNA in the skin had CuV IgG-antibodies already 5-21 years before the skin biopsies were taken. This suggests that CuV can persist for decades after primary infection similarly to human parvovirus B19. However, even if some preliminary disease associations exist, the role of CuV in CTCL or other skin cancers needs further investigations. TuV findings, both DNA and antibodies, were absent or scarce. TuV DNA was not detected in any of the skin tissues, and TuV IgG was detected only in one child and one transplant patient among the serum samples of 1500 individuals ranging four continents. Overall, more studies are needed to confirm if TuV truly is a human virus or just accidentally occurring in human samples.