Gene therapy has received much attention the last decade as a method to correct a number of disorders arising from a defective gene. Gene therapy can be defined as the introduction of a functional genetic element into a cell for a therapeutic purpose. This is a very broad term and gene therapy can be applied to a wide range of diseases from genetic diseases such as cystic fibrosis to infectious diseases or even acquired genetic diseases such as cancers. Adenoviruses (Ad) are the second most common vector for gene therapy in clinical trials today, and these vectors are mostly based on serotype 2 or 5 (Ad2 and Ad5). It has been shown that Ad2 and Ad5 use a receptor that is often downregulated in malignant cells and they also suffer from shortcomings because of the high levels of pre-existing immunity against these serotypes in the society. Hence, new and improved vectors serving as alternatives to these serotypes need to be developed to make gene therapy a successful treatment option. The work presented herein is devoted to analyse what alternative adenovirus vectors could be used for treatment of glioma brain tumours. A number of different adenovirus serotypes were screened for their ability to infect human glioma tumour cells in vitro. Established cell lines as well as low-passage glioma cells from different donors were used. Adenovirus serotype 11p (Ad11p) proved to be a promising vector candidate because of its capacity to efficiently infect the glioma cells and its low prevalence in the society. The complete genome of this serotype was sequenced to further develop this as an alternative adenovirus vector. Furthermore, a number of cell lines were produced to generate E1 deleted Ad11p vectors. Other promising vector candidates were Ad16 and a chimpanzee adenovirus called CV23. Ad16 was the most efficient human serotype to infect human low-passage glioma cells and the prevalence for this serotype is also very low. The overall most efficient virus was surprisingly the non-human CV23 virus. This adenovirus has no prevalence in humans, but efficiently infects human cells in vitro. The first analysis was made on established glioma cell lines and was followed up by using low passage glioma cells from a number of different patients. The glioma cells were analysed when subjected to <20 passages (low passage) and then again at >40 passages (high passage). The cells at a higher passage number were significantly more permissive to Ad5 than the cells analysed at a low passage number. This could in part explain why some of the promising in vitro data for Ad5 have shown a limited success in vivo. In contrast, CV23 infected the low and high passage gliomas equally. This indicates that CV23 uses an internalisation mechanism subjected to less variation than the mechanism used by Ad5. We further characterised the receptor interaction of CV23 and found that none of the previously known primary receptors for adenoviruses were of importance for binding. We found that bovine serum albumin present in the growth medium was responsible for the high binding capacity to cells. Binding is a criterion for the first step of the infection, but not necessarily a good correlate to the infection capacity. CV23 infected human cells efficiently also in the absence of bovine serum albumin.

Influenza virus is a RNA virus that exists as different types and subtypes. Influenza A virus strains are known to cause disease in several bird and mammalian species. Wild birds are believed to constitute the natural reservoir for influenza A virus. In humans, influenza A virus causes yearly seasonal influenza epidemics of respiratory disease resulting in high morbidity and severe economic consequences. Due to the virus’ ability to change its antigenic properties by mutation, yearly vaccination is required for protection from the disease. There are many different subtypes of influenza virus which are characterized according to two surface structures - the hemagglutinin and neuraminidase proteins - , for example; H5N1. These subtypes have the ability to recombine, and thereby creating new variant combinations. If a subtype that the living population of humans has not encountered before starts to spread among humans, it can result in a pandemic. Pandemic outbreaks have occurred at irregular intervals throughout history and have had a devastating impact on mankind. For example the Spanish influenza pandemic of 1918 is thought to have killed more than 50 million people. Influenza A virus is also an important cause of disease in poultry where virus strains of some subtypes may change into forms that are highly pathogenic. These virus strains may transmit directly to man and multiple other species. This has been the case in the ongoing outbreak that started in South East Asia in 2003. All known subtypes of influenza A virus have been isolated from wild birds living in aquatic environments, mainly dabbling ducks. These species are considered to be the reservoir for influenza A virus. The virus causes sub clinical gastrointestinal infection in ducks. High amounts of virus are excreted in the feces and spread via the fecal-oral route through water where it can persist for a prolonged time. There are still many unknowns about the ecology of influenza virus in the wild bird reservoir. This thesis includes five articles where data are presented that add new knowledge on this subject. We add proof that wild ducks are indeed the host for most influenza A virus subtypes by presenting data from a meta-analysis on all published screening data from wild birds and by presenting data from a four year screening of migratory ducks that were caught and sampled at Ottenby Bird Observatory. Our investigations have shown that the prevalence of influenza virus in the wild duck population of western Eurasia shows temporal differences in comparison to the results found in studies in North America. The prevalence in western Eurasian ducks is high during the period August to December and also rises in the spring. These findings are of importance for the understanding of how influenza virus is perpetuated in nature. During the course of the study only low pathogenic subtypes were isolated. Of concern is the high frequency of isolation of virus strains of the H5 and H7 subtypes that are prone to change into highly pathogenic variants in poultry. Many of the strains isolated in our study are similar to the ones that have caused influenza outbreaks in poultry in Europe during the last seven years. This indicates that wild bird surveillance for influenza A virus can be of major value as a sentinel system to prevent outbreaks in domestic poultry. Studies on Black-headed Gulls (Larus ridibundus) revealed a previously unknown subtype, H16. This finding widened the spectra of known influenza A virus subtypes in nature. Influenza A virus was also isolated in samples from Guillemots (Uria aalge) in the Baltic Sea. This was the first time influenza A virus was isolated from this species in Europe. The isolated virus strains contained a mix of genes, some of which must have been derived from influenza A virus strains present in the North American bird population. This finding proves that limited exchanges between the virus strains present on the American and the Eurasian continents exist, which is of concern for evaluating the risk of spread of highly pathogenic virus strains by wild birds to the Americas. / Influensavirus är RNA virus och indelas i olika typer och subtyper. Influensa A virus orsakar sjukdom hos ett flertal fågel- och däggdjursarter. Vilda fåglar anses utgöra den viktigaste reservoaren för influensa A virus. Hos människa orsakar influensa A virus årliga epidemier av luftvägssjukdom med hög sjuklighet och stora ekonomiska konsekvenser för samhället. Eftersom frekventa mutationer orsakar ändringar i virusets ytstrukturer krävs årlig vaccination med nytt anpassat vaccin för att ge skydd mot sjukdom. Det finns många olika subtyper av influensa A virus. Dessa karaktäriseras med två av virusets ytstrukturer; hemagglutinin och neuraminidas, vilket till exempel skrivs H5N1. Virus av olika subtyper kan rekombinera och på så sätt skapa nya varianter. Om en subtyp som tidigare ej cirkulerat bland världens befolkning orsakar ett utbrott kan detta leda till en världsomfattande epidemi, en så kallad pandemi. Pandemier har drabbat mänskligheten med viss regelbundenhet genom historien och haft förödande konsekvenser. Till exempel orsakade pandemin ”Spanska sjukan” under åren 1918-1920 mer än 50 miljoner dödsfall. Influensa A virus orsakar också förödande utbrott i fjäderfäbesättningar. Virus av vissa subtyper kan mutera till högpatogena varianter och orsaka så kallad högpatogen aviär influensa. Dessa högpatogena varianter kan även överföras till och orsaka sjukdom hos människa och andra djur vilket varit fallet under det pågående utbrott av H5N1 som startade i sydöstra Asien 2003. Alla kända subtyper av influensa A virus har isolerats i material från vilda fåglar vilka lever i vattenmiljö, framförallt från änder. Dessa arter anses därför utgöra influensavirusets reservoar i naturen. Hos änder orsakar viruset framförallt en subklinisk infektion i gastrointestinalkanalen och sprids genom faekal-oral överföring via vatten i vilket viruset kan förbli aktivt en längre tid. Det finns fortfarande många obesvarade frågor angående influensa A virus ekologi bland vilda fåglar. I denna avhandling presenteras fem artiklar som tillför ny kunskap inom detta område. I avhandlingen styrks bevisen för att vilda änder utgör virusets reservoar i naturen dels genom en metaanalys av samtliga publicerade data rörande fynd av influensa A virus hos vilda fåglar, dels med hjälp av data från fyra års provtagning från flyttande vilda änder vid Ottenby fågelstation. Resultaten påvisar temporala skillnader i influensvirusets prevalens i den västeuroasiatiska andpopulationen jämfört med den nordamerikanska. Prevalensen i den västeuroasiatiska andpopulationen är hög under perioden augusti till december och i viss mån även under våren. Dessa fynd talar för att influensavirus kontinuerligt cirkulerar i andpopulationen. Under studien av förekomsten av influensa A virus hos änder isolerades enbart olika lågpatogena subtyper. Subtyperna H5 och H7 var vanligt förekommande. Dessa subtyper är benägna att utvecklas till högpatogena varianter om de sprids till fjäderfäbesättningar med svåra konsekvenser som följd. Genom studier av virus släktskap visas att de virus vi isolerat från vilda änder är snarlika de som orsakat utbrott bland fjäderfä i Europa under de senaste sju åren. Detta styrker värdet av att övervaka förekomsten av influensavirus hos vilda fåglar för att på så sätt förhindra utbrott av sjukdom bland fjäderfä. Undersökning av prover från skrattmås (Larus ridibundus) ledde fram till upptäckten av en helt ny subtyp av influensavirus; H16. Därmed utvidgades spektret av kända subtyper i naturen. Influensa A virus isolerades från sillgrisslor (Uria aalge) i Östersjön vilket inte tidigare gjorts hos denna art i Europa. Dessa virus innehöll gener från både nordamerikanska och euroasiatiska fågelpopulationers virus. Det visar att det finns ett utbyte av virus mellan fågelpopulationerna på de skilda kontinenterna. / On the day of the defence data the status on article IV was Submitted and the title was "Multi-year surveillance of influenza virus type A in migratory waterfowl in northern Europe".

Adenoviruses are common pathogens all over the world. The majority of the population has at some point been infected with an adenovirus. Although severe disease can occur in otherwise healthy individuals an adenovirus infection is most commonly self limited in these cases. For immunocompromised individuals however, adenoviruses can be life-threatening pathogens capable of causing disseminated disease and multiple organ failure. Still there is no approved drug specific for treatment of adenovirus infections. We have addressed this using a unique whole cell viral replication reporter gene assay to screen small organic molecules for anti-adenoviral effect. This RCAd11pGFP-vector based assay allowed screening without any preconceived idea of the mechanism for adenovirus inhibition. As a result of the screening campaign 2-[[2-(benzoylamino)benzoyl]amino]-benzoic acid turned out to be a potent inhibitor of adenoviral replication. To establish a structure-activity relationship a number of analogs were synthesized and evaluated for their anti-adenoviral effect. The carboxylic acid moiety of the molecule was important for efficient inhibition of adenovirus replication. There are 54 adenovirus types characterized today and these are divided into seven species, A-G. The receptors used by species B and other adenoviruses are not fully characterized. CD46 is a complement regulatory molecule suggested to be used by all species B types and some species D types but this is not established. We have designed a new bioassay for assessment of the interaction between adenoviruses and CD46 and investigated the CD46-binding capacity of adenovirus types indicated to interact with CD46. We concluded that Ad11p, Ad34, Ad35, and Ad50 clearly bind CD46 specifically, whereas Ad3p, Ad7p, Ad14, and Ad37 do not. CD46 is expressed on all human nucleated cells and serves as a receptor for a number of different bacteria and viruses. Downregulation of CD46 on the cell surface occurs upon binding by some of these pathogens. We show that early in infection Ad11p virions downregulate CD46 upon binding to a much higher extent than the complement regulatory molecules CD55 and CD59. These findings may lead to a better understanding of the pathogenesis of adenoviruses in general and species B adenoviruses in particular and hopefully we have discovered a molecule that can be the basis for development of new anti-adenoviral drugs.

Viral infections are constantly increasing and impose a large threat to the public health. Alphaviruses are responsible for several animal and human diseases and have a large medical importance with few treatments available today. Alphaviruses are small, spherical single stranded RNA viruses, and are most often transmitted by mosquito vectors. Alphaviruses contains a domain of nonstructural proteins that compose the replication machinery. The domain is crucial for viral replication to occur and is therefore an interesting target for antiviral therapy. With the focus on Chikungunya and Semliki Forest Virus this work investigates the events in the cells on molecular level during infections. To do this a panel of Camelid derived single domain antibodies are developed to target the nonstructural proteins of Chikungunya and Semliki Forest Virus. Binding of the produced nanobodies to the viral proteins was investigated by biochemical methods including immunoprecipitations, western blot, and ELISA. Cell lines that express nsP-specific nanobodies in the cytosol were employed for infection- and plaque assays with Semliki Forest Virus in order to determine the antiviral potential of the new nanobodies. Three of the nanobodies proved to bind two different nonstructural proteins of the viruses, providing opportunities for further investigations and a possible use of these nanobodies to identify viral vulnerabilities that could be exploited for antiviral intervention.

Syftet med denna uppsats är att bidra till en ökad förståelse av elevers kunskaper om och intresse för virologi. För att nå syftet utfördes en kvantitativ enkätundersökning riktad till elever i grundskolan årskurs 9 och årskurs 3 i naturvetenskapsprogrammet. Resultaten visade att eleverna på naturvetenskapsprogrammet har en högre kunskapsnivå i virologi och ett självskattat större intresse i virologi än eleverna i årskurs 9. Studien visar även på att eleverna anser familj och vänner och digitala medier som viktiga källor till deras kunskaper i virologi. Delar av svarsdata indikerade att nyhetsrapporteringen om sars-cov-2/covid-19 har påverkat elevernas kunskaper inom ämnesområden som virusstruktur och virussjukdomar. Resultaten tolkas spegla att eleverna på naturvetenskapsprogrammet utgör ett äldre och mer studieintresserat urval än eleverna i årskurs 9. Studien kan bidra till en förbättrad virologiundervisning genom att bistå med underlag till diagnostisk bedömning av elevers kunskaper och till konstruerandet av samhällsfrågor med naturvetenskapligt innehåll.

Cervical cancer is the second most common cancer among women worldwide and human papillomavirus (HPV) is a prerequisit for the development of this cancer. HPV belongs to the Papillomaviridae family and infects the basal layer of epithelial cells where it generally progresses into warts or condylomas. HPV can only reproduce in differentiating epithelia and it is therefore difficult to study the natural infection of HPV. More than 100 HPV types exist and they are divided into different genera based on their L1 open reading frame sequence. Most of the HPV types in the alpha-papillomavirus genus infect the mucosal epithelium while HPVs from the beta-papillomavirus genus usually infect cutaneous epithelial cells. Presently, it is not known what decides the anatomical tropism and our aim was to study determinants of this tropism. By using HPV virus like particles (VLP) and pseudovirus we found that VLPs from the two alpha-papillomaviruses HPV-6 and HPV-16 interacted with cell-surface heparan sulfate (HS) for initial attachment. When we labelled HPV VLPs with a fluorescent dye to study internalization HPV-6 was more strongly inhibited than HPV-16. Furthermore, a pseudovirus infection assay demonstrated that the beta-papillomavirus HPV-5 was less dependent on HS for infection than HPV-16. By analyzing the isoelectric point (p1) of the HPV L1 capsid protein we found that alpha HPV types were more positively charged than beta HPV types. Also, HPV-6 had a higher positive charge than HPV-16. Thus, the inhibition of the negatively charged heparin against HPV infection was clearly related to the charge of the HPV L1 capsid. This suggested that the initial interaction could be one of the determinants of tropism although not the sole factor. Lactoferrin is a protein found in milk, saliva, semen, tear fluid and endocervical secretions that has antiviral activities. Both human and bovine lactoferrin inhibited HPV infection but we found no significant differences in inhibition of alpha- and beta-papillomavirus infection. We could however demonstrate that different lactoferricins, small peptide derivates from the N-terminal part of lactoferrin, were able to inhibit HPV infection. This antiviral activity depended on lactoferricin peptide, HPV type and cell origin. The regulation of HPV gene expression in the host cell could also determine HPV tropism. The HPV long control region (LCR) contains cis-responsive elements that regulate HPV transcription and the epithelial tropism of HPV is determined by epithelial specific constitutive enhancers in the LCR. It has been hypothesized that the combination of transcription factors in the host cell determines the cell-type-specific expression. In cells with a skin origin the HPV-5 LCR was twice as efficient in transcriptional activation compared to HPV-16 LCR, while in cervical cells the HPV-16 LCR was almost twice as effective in activating transcription compared to HPV-5 LCR. To conclude, alpha- and beta-papillomaviruses differed regarding their ability to infect cells and regulate viral gene expression. These abilities corresponded with their natural host cells and suggested that HPV anatomical tropism could be determined at several steps in the HPV life cycle.

Adenoviruses belong to the most common human pathogens. The severity of infection varies greatly, from subclinical to lethal, depending on the virus type and immune status of the infected host. The 51 known human adenovirus serotypes are divided into six species (A-F) based on characteristics such as tropism. Species B adenoviruses, which are the subjects of this thesis, are further divided into subspecies B:1 that contains Ad3, Ad7, Ad16, Ad21 and Ad50 and subspecies B:2 that contains Ad11, Ad14, Ad34 and Ad35. Species B adenoviruses primarily cause ocular and respiratory tract infections, but certain serotypes (Ad11, Ad34 and Ad35) are also associated with renal disease. The main aim of this thesis was to identify and characterize cellular receptors for species B adenoviruses. This will ultimately help to understand the diverse tropism shown by different adenoviruses and perhaps contribute to development of antivirals. Also, since adenoviruses are among the most commonly used vector for gene therapy it is of importance to characterize the initial steps of adenovirus life cycle. Members of species B adenoviruses have been shown to utilize both the complement regulating membrane cofactor protein (MCP), i.e. CD46, and a still unknown receptor. CD80 and CD86, usually found on antigen-presenting cells, have also been suggested as receptors We found first that Ad11 used CD46 as a cellular receptor on respiratory A549 cells, and subsequently that CD46 is a cellular receptor for all species B adenovirus serotypes, except for adenovirus types 3 and 7, using cells that represent the tropism of species B adenoviruses, i.e. respiratory, conjunctival and renal epithelial cells. We further compared the relative roles of CD46 with CD80 and CD86 using cells that represent species B adenovirus tropism. Using soluble candidate receptors and antibodies against corresponding receptors to challenge virus binding to and infection of cells, we found that on these cells, CD46 is a cellular receptor for all species B adenoviruses except Ad3 and Ad7, and that CD80 and CD86 do not play an important role. We have further pinpointed the interaction site for Ad11 on CD46 by X-ray crystallography. The extracellular region of CD46 contains four short consensus repeats (SCR1-4) of which the outermost N-terminal SCR1 and SCR2 mediate binding to Ad11. This interaction was confirmed by inhibiting infection and binding of Ad11 to A549 cells using soluble SCR1-2 fragments. Surprisingly the conformation of bound CD46 differs profoundly from its unbound state, with the bent surface structure straightened into an elongated rod. Viral proteins can sometimes undergo large conformational changes upon receptor binding, but this is, to the best of our knowledge, the first example of a virus protein dramatically changing the overall structure of its receptor. CD46 serves as a receptor for a large number of viral and bacterial pathogens and it is structurally and functionally related to other viral receptors such as CD21 and CD55. The mode of interaction presented here may serve as a conceptual framework for studies of many other receptors that are constructed from SCR domains.

Diss. (sammanfattning) Uppsala : Uppsala universitet, 2007. / Härtill 6 uppsatser.

Hantaviruses are found practically all over the world and cause hemorrhagic fevers in man. Each year about 150,000 people are hospitalized in these zoonotic infections which can be of two types: hemorrhagic fever with renal syndrome (HFRS) or hantavirus cardiopulmonary syndrome (HCPS), depending on the infecting virus. Hantavirus infections are emerging infectious diseases. That is, the number of reported cases of hantaviral disease is increasing, new hantaviruses are discovered continually, and already known hantaviruses are expected to spread to new areas. Therefore, knowledge and monitoring of these viruses are imperative from a public health perspective. In this thesis, the characterization of a local human Puumala (PUUV) virus isolate is described. Genetic and serological relationships to other hantaviruses are investigated and the viral protein interactions, critical for genome packaging and assembly, are studied. We found that the nucleotide and amino acid sequences of the local PUUV strains are significantly different from the PUUV prototype strain Sotkamo, a difference that indicates that there might be a risk of misdiagnosing PUUV infected patients when using reagents derived from the prototype strain. These data contributed to the introduction of locally derived diagnostic tools to the Laboratory of Clinical Virology at the Umeå University hospital, which is the reference centre for hantaviral diseases in Sweden. Furthermore, when studying the underlying mechanisms of genome packaging, we identified several regions and amino acids absolutely required for nucleocapsid protein interactions. Also, a region that appeared to regulate this interaction was discovered. Finally, the serological immune responses in DNA-vaccinated mice and PUUV infected patients were investigated. We found that the cross-reactive antibody response in vaccinated mice and in infected individuals was unique and independent of homologous titres. Furthermore, four immunodominant epitopes with specific cross-reactive characteristics were identified. Our findings have highlighted the complexity of the serological immune responses to hantavirus infections, and they emphasize the importance of customizing the diagnostic tools and performing clinical analyses on locally derived strains. In conclusion, we believe that these results are valuable in the development of new serological, genetic, and epidemiological tools.

It has been suggested that the rising amounts of chemicals in the environment may affect host resistance and increase susceptibility to infections. Studies have also shown that infections can change the toxicity of pollutants. The aim of this thesis was to study interactions between environmental pollutant exposure in terms of polybrominated diphenyl ethers (PBDE) and a common human coxsackievirus B3 (CVB3) infection adapted to Balb/c mice. The studies focused on virus levels, cytokines, metabolising cytochrome P450 (CYP) enzymes and tissue distribution of PBDE. A novel finding was an organ-specific effect of CVB3 infection on the metabolising capacity of PBDE. The PBDE metabolising enzyme CYP2B10 was down-regulated by the CVB3 infection in the liver, up-regulated in the lungs, but not affected in the pancreas. Accordingly, CVB3 infection increased the concentration of PBDE in the livers of infected mice. However, serum levels of PBDE were not affected by the infection, indicating that serum does not reflect the actual organ exposure of PBDE in infected individuals. The change in metabolising capacity was likely mediated by infection-induced cytokines and associated effects on the nuclear factor-κB (NF-κB) pathway. PBDE drastically decreased serum levels of several cytokines and chemokines, an event that may create a slot for viruses to replicate. Accordingly, some results show that infected mice exposed to a high dose of PBDE had higher virus levels than mice exposed to a low dose. In conclusion, infected individuals showed organ-specific changes in metabolism and tissue levels of PBDE, which potentially could change the toxicity of PBDE. PBDE also seems to affect the fate of the infection. NF-κB activated pathways may mediate one possible mechanism underlying these effects. Thus, further investigations of this pathway are warranted. In addition, future studies should address how PBDE exposure affects viral replication.