To date, the use of mitochondrial DNA in forensic analysis has relied on the presence of variations in the control region to differentiate between samples. One problem that this analysis has shown is the occurrence of common Haplogroup H haplotypes or identical sequences. Thus, there is a need to enhance the distinguishing power of this type of analysis. One option has been to investigate the mitochondrial coding region for polymorphisms that could differentiate between samples with identical control region haplotypes. The goal of this study has been to identify polymorphic coding region sites for development in a Pyrosequencing assay that would effectively enhance the discriminatory power of mitochondrial DNA analysis. With this goal in mind, five duplexes have been successfully developed and tested, utilizing the ten polymorphic sites that had been selected, with most sites being specific to Caucasians. Validation studies were performed to test the durability of the assay. The specificity of the assay to primate and non-primate species was determined to be limited to primate species only. Sample variations, including mixtures, dilutions and environmental exposure, were utilized to assess the sensitivity of the Pyrosequencing method. It was found that a minimum initial DNA input of 10fg was necessary for reliable results. The Pyrosequencing assay was able to detect mixtures at a 1:1 ratio and environmental samples exposed to the elements from up to 1 week for blood and 6 weeks for semen. Samples designed to simulate typical casework materials were analyzed and found to provide for consistent results, including trace fingerprints and digested hair shafts. These validation results provide the conclusion that this assay is suitable for use in forensic casework and demonstrate that the mitochondrial coding region provides a viable alternative to hypervariable region analysis.

No description available.

Neuraminidase (NA) is one of two major antigens on the surface of influenza A viruses. It is comprised of a single N-terminal transmembrane domain (TMD), a stalk domain, and a C-terminal enzymatic head domain that cleaves sialic acid, most notably to release new particles from the host cell surface. NA is only enzymatically active as a homo-tetramer. However, it is not known which properties facilitate the oligomerization of NA during assembly. Our results show that, apart from anchoring the protein to the membrane, the NA TMD also contributes to the assembly process by keeping the stalk in a tetrameric conformation. The ability of the TMD to oligomerize is shown to be dependent on its amphiphilic characteristics that was largely conserved across the nine NA subtypes (N1-N9). Over time the NA TMDs in human H1N1 viruses were found to have become more amphiphilic, which correlated with stronger oligomerization. An old H1N1 virus with a more recent N1 TMD had impaired growth, but readily acquired compensatory mutations in the TMD to restore growth, by reverting the TMD oligomerization strength back to that of the old TMD, demonstrating a biological role of the TMD in folding and assembly. NA and the other viral proteins are spatially and temporally coordinated to achieve optimal viral production. By using a co-transfection analysis, the high AU-content in the NA and HA ER-targeting sequence coding regions (for NA TMD as well as the HA signal sequence) were found to inhibit their expression. The inhibition was alleviated by the early expressed influenza RNA-binding protein NS1, which promoted translation and showed enriched foci at the endoplasmic reticulum (ER). NS1, which expresses early during infection, is therefore likely the regulator of NA and HA to prevent premature expression. These results show that the NA TMD is under substantial selection pressure at both the nucleotide and amino acid level to accommodate its roles in ER-targeting, protein folding, and post-transcriptional regulation. / <p>At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 4: Accepted.</p>

Vector-borne diseases are an increasing global threat to humans due to climate changes, elevating the risk of infections transmitted by mosquitos, ticks, and other arthropod vectors. Ixodes ricinus, a common tick in Europe, transmits dangerous tick-borne pathogens to humans. Tick-borne encephalitis (TBE) is a vector-borne disease caused by TBE virus (TBEV). Climate change has contributed to increased tick abundance and incidence of tick-borne diseases, and between 10,000 and 15,000 human TBE cases are reported annually in Europe and Asia. TBEV shows a patchy geographical distribution pattern where each patch represents a natural focus. In nature, TBEV is maintained within the tick-rodent enzootic cycle. Co-feeding is the main route for TBEV transmission from infected to uninfected ticks and for maintenance within the natural foci. The increasing number of TBE cases in Scandinavia highlights the importance of characterizing additional TBEV sequences and of identifying novel natural foci, and in this work we sequenced and phylogenetically characterized four TBEV strains: Saringe-2009 (from a blood-fed nymph), JP-296 (from a questing adult male), JP-554 (from a questing adult male), and Mandal-2009 (from a pool of questing nymphs, n = 10). Mandal-2009 represents a TBEV genome from a natural focus in southern Norway. Saringe-2009 is from a natural endemic focus in northern Stockholm, Sweden, and JP-296 and JP-554 originate from a natural focus “Torö” in southern Stockholm. In addition, we have studied the effect of different biotic and abiotic factors on population dynamics of I. ricinus in southern Stockholm and observed significant spatiotemporal variations in tick activity patterns. Seasonal synchrony of immature stages and total tick abundance are important factors for the probability of horizontal transmission of TBEV among co-feeding ticks. We found that the probability of co-occurrence of larvae, nymphs, and female adults was highest during early summer whereas increasing vegetation height and increasing amounts of forest and open water around the study sites had a significant negative effect on co-occurrence of larvae, nymphs, and female adults. The proximal part of the 3 ́non-coding region (3 ́NCR) of TBEV contains an internal poly(A) tract, and genomic analysis of Saringe-2009 revealed variability in the poly(A) tract indicating the existence of different variants within the TBEV pool of Saringe-2009. Like other RNA viruses, TBEV exists as swarms of unique variants called quasispecies. Because Saringe-2009 came from an engorged nymph that had been feeding on blood for &gt;60 h, we propose that Saringe-2009 represents a putative shift in the TBEV pool when the virus switches from ectothermic/tick to endothermic/mammalian environments. We investigated the role of poly(A) tract variability in replication and virulence of TBEV by generating two infectious clones of the TBEV strain Toro-2003, one with a short/wild-type (A)3C(A)6 poly(A) tract and one with a long (A)3C(A)38 poly(A) tract. The infectious clone with the long poly(A) tract showed poor replication in cell culture but was more virulent in C57BL/6 mice than the wild-type clone. RNA folding predictions of the TBEV genomes suggested that insertion of a long poly(A) tract abolishes a stem loop structure at the beginning of the 3 ́NCR. Next generation sequencing (NGS) analysis of the TBEV genomes after passaging in cell culture and/or mouse brain revealed molecular determinants and quasispecies structure that might contribute to the observed differences in virulence. Our findings suggest that the long poly(A) tract imparts instability to the TBEV genome resulting in higher quasispecies diversity that in turn contributes to TBEV virulence. Phylogenetic analysis of Saringe-2009, JP-296, JP-554, and Mandal-2009 predicted a strong evolutionary relationship among the four strains. They clustered with Toro-2003, the first TBEV strain from Torö, demonstrating a Scandinavian clade. Except for the proximal part of the 3 ́NCR, TBEV is highly conserved in its genomic structure. Genomic analysis revealed that Mandal-2009 contains a truncated 3 ́NCR similar to the highly virulent strain Hypr, whereas JP-296 and JP-554 have a genomic organization identical to Toro-2003, the prototypic TBEV strain from the same natural focus. NGS revealed significantly higher quasispecies diversity for JP-296 and JP-554 compared to Mandal-2009. In addition, single nucleotide polymerphism (SNP) analysis showed that 40% of the SNPs were common between quasispecies populations of JP-296 and JP-554, indicating the persistence and maintenance of TBEV quasispecies within the natural focus. Taken together, these findings indicate the importance of environmental factors for the occurrence pattern of the different life-stages of the tick vector, which are important for the persistence of TBEV in nature. Our findings also show that the selection pressure exerted by specific host also affects the population structure of the TBEV quasispecies. In addition, our results further demonstrate that the evolution of quasispecies has effect on TBEV virulence in mice. / Vektorburna sjukdomar är ett växande globalt hot mot både människor och djur. De pågående klimatförändringarna kan leda till förhöjda risker för infektioner överförda av myggor, fästingar och andra leddjursvektorer. Ixodes ricinus är en vanlig fästing i Europa som överför fästingburna patogener som är farliga för människor. Fästingburen encefalit (TBE) är en vektorburen sjukdom som orsakas av TBE-virus (TBEV). De pågående klimatförändringarna har bidragit till en ökning både av vektorn och sjukdomsfrekvensen. Mellan 10 000 och 15 000 mänskliga TBE-fall rapporteras årligen i Europa och Asien. Den geografiska fördelningen av TBEV visar ett ojämnt fördelningsmönster där viruset är koncentrerat till vissa fokusområden. TBEV återfinns i naturen i en livscykel där viruset hela tiden överförs mellan fästingar och däggdjur. Spridningen sker dels från en infekterad fästing till ett ryggradsdjur när fästingen äter på värddjuret. Spridning mellan fästingar sker troligen främst genom så kallad “co-feeding”, det vill säga att flera fästingar suger blod samtidigt från samma värddjur. Viruset kan då passera från en infekterad fästing, genom värddjuret till oinfekterade fästingar. Virus kan identifieras och studeras med genetiska metoder. Det ökande antalet TBE-fall i Skandinavien styrker vikten av att hitta och karakterisera ytterligare TBEV-stammar och identifiera nya naturliga fokusområden. Vi har sekvenserat och fylogenetiskt beskrivit fyra TBEV-stammar: Saringe-2009 (blodfylld nymf), JP-296 (födosökande vuxen hane), JP-554 (födosökande vuxen hane) och Mandal-2009 (födosökande nymfer, n = 10). Mandal-2009 är ett TBEV från ett naturligt fokusområde i södra Norge. Saringe-2009 kommer från ett naturligt fokusområde i norra Stockholms län, Sverige. JP-296 och JP-554 härstammar från Torö som är ett naturligt fokusområde i södra Stockholms län, Sverige. Förutom den genetiska sekvenseringen av TBEV har vi också studerat effekten av olika biotiska och abiotiska faktorer på populationsdynamik av I. ricinus i södra Stockholm och observerade variation i fästingsaktivitetsmönster både temporalt och spatialt. Förekomstmönster av fästinglarver, nymfer och vuxna honor, och det totala antalet fästingar är viktiga faktorer för sannolikheten för horisontell överföring av TBEV mellan fästingar. Vi fann att sannolikheten för synkron förekomst av larver, nymfer och honor var högst under försommaren. Vegetationshöjd, mängden skog och mängd öppet vatten runt undersökningsområden hade signifikanta negativa effekter på sannolikheten för att larver, nymfer och honor skulle förekomma samtidigt. Den variabla delen av den icke-kodande 3 ́regionen (3'NCR) av TBEV-genomet innehåller ofta en intern poly(A)-sekvens. Liksom andra RNA-virus, förekommer TBEV som så kallade ”quasispecies” vilka definieras som grupper av olika genetiska varianter av virus. Genom analysen av TBEV-stam Saringe-2009 avslöjades variation i poly(A)-sekvensen vilket indikerar förekomst av ”quasispecies”. Eftersom Saringe-2009 kom från en blodfylld nymf som hade sugit blod i &gt; 60 timmar, föreslår vi att Saringe-2009 visar en förändring i ”quasispecies”-poolen när viruset överförs från exoterm fästingmiljö till endoterm däggdjursmiljö. Vi undersökte poly(A)-ekvensens variabilitet och dess roll vid replikering och för virulens hos TBEV, genom att skapa två infektiösa kloner av Torö-2003 stammen; en med en kort/vild-typ (A)3C(A)6 poly(A)-sekkvens, och en med en lång (A)3C(A)38 poly(A)-sekvens. Den infektiösa klonen med lång poly(A)-sekvens replikerade sämre än vildtypklonen i cellkultur, men (A)3C(A)38 poly(A) var mer virulent i C57BL/6-möss än (A)3C(A)6 poly(A). Datasimulering av TBEV-genomets sekundär-RNA-struktur visade att de längre poly(A)-sekvenserna påverkar veckningen av en specifik sekundärstruktur (SL14) i början av 3 ́NCR. Djupsekvenseringsanalys av TBEV-gnomen avslöjade skillnader för specifika gener och ”quasispecies”-strukturen efter passering i cellkultur och/eller mushjärna. Dessa förändringar föreslås bidra till de observerade skillnaderna i virulens. Våra resultat indikerar att den långa poly(A)-sekvensen ger instabilitet i TBEV-genomet, vilket resulterar i ökad mångfald av ”quasispecies”-populationen som i sin tur kan bidra till TBEV-virulens. Fylogenetisk analys av Saringe-2009, JP-296, JP-554 och Mandal-2009 visade på ett nära släktskap mellan de fyra skandinaviska TBEV-stammarna. De nya stammarna formerade ett kluster med en tidigare TBEV-stam identifierad på Torö (Toro-2003), vilket skapade ett skandinaviskt klad. Genetisk analys visade att Mandal-2009 innehåller en trunkerad 3 ́NCR som liknar den högvirulenta stammen HYPR. JP-296 och JP-554 hade däremot samma genetiska struktur som den längre Torö-2003 stammen från samma fokusområde. Djupsekvensering visade höge mångfald av ”quasispecies”-populationen för JP-296 och JP- 554 jämfört med Mandal-2009. Analys av enkel nukleotid polymorfism (SNP) visade att 40 % av alla SNP var gemensamma mellan ”quasispecies”-populationen för JP-296 och JP-554. Detta indikerar att TBEV-”quasispecies”-strukturen kan vara konserverad för närbesläktade virus vilken kan leda till att den bevaras inom specifika fokusområden. Sammantaget så visar dessa studier att miljöfaktorer påverkar förekomsten av fästingvektorn och dess olika livsstadier, vilket är en bakomliggande faktor för utbredning av TBEV i naturliga fokusområden. Det visar även på att värdmiljön påverkar strukturen för ”quasispecies”-populationen. Dessutom visar våra studier att evolution och utveckling av ”quasispecies”-strukturen kan påverka virulensen för TBEV i möss.

<p>Genetic profiling is commonly performed on the autosomes using multiple DNA markers. Although routine forensic DNA analysis is robust and based on reliable technologies, samples with degraded or limited amounts of DNA often fail. In these cases, the analysis of mitochondrial DNA (mtDNA) can be very valuable due to the high copy number per cell. This thesis describes evaluation and modifications of existing technologies that are useful in forensic DNA typing, mainly focusing on mtDNA.</p><p>DNA quantities isolated from common evidence materials such as hairs, fingerprints and accessories were estimated using a real-time quantification assay. Knowledge of quantitative differences between materials can guide forensic scientists to perform the best analysis (Paper I).</p><p>The current mtDNA analysis is based on hypervariable region (HVI/HVII) sequencing, which is the most rigorous and time-consuming forensic DNA analysis. Therefore, we evaluated the possibility to exclude individuals by screening for non-matching samples using the rapid and easy mtDNA Linear Array Assay (Paper II). </p><p>The major disadvantage using mtDNA is the lower discrimination power compared to multiple nuclear DNA markers. In contrast to the nuclear genome, due to the uniparental (maternal) mode of inheritance, no individual has unique mtDNA. We investigated the possibility of increasing the discrimination power by using pyrosequencing technology to analyse parts of the coding region in addition to HVI/HVII (Paper III). Furthermore, the addition of coding mtDNA information was evaluated by comparing several recently published mtDNA coding region assays (Paper IV). </p><p>Mixtures of DNA are common in forensic genetics due to contribution of DNA from several individuals, contamination or heteroplasmy. To resolve mixtures we have developed a pyrosequencing-based assay for the accurate quantification of the mtDNA mixture components (Paper V).</p><p>In conclusion, this thesis describes several assays that are valuable in forensic genetics for DNA quantification, improved mtDNA analysis, and mtDNA mixture interpretation.</p>

Genetic profiling is commonly performed on the autosomes using multiple DNA markers. Although routine forensic DNA analysis is robust and based on reliable technologies, samples with degraded or limited amounts of DNA often fail. In these cases, the analysis of mitochondrial DNA (mtDNA) can be very valuable due to the high copy number per cell. This thesis describes evaluation and modifications of existing technologies that are useful in forensic DNA typing, mainly focusing on mtDNA. DNA quantities isolated from common evidence materials such as hairs, fingerprints and accessories were estimated using a real-time quantification assay. Knowledge of quantitative differences between materials can guide forensic scientists to perform the best analysis (Paper I). The current mtDNA analysis is based on hypervariable region (HVI/HVII) sequencing, which is the most rigorous and time-consuming forensic DNA analysis. Therefore, we evaluated the possibility to exclude individuals by screening for non-matching samples using the rapid and easy mtDNA Linear Array Assay (Paper II). The major disadvantage using mtDNA is the lower discrimination power compared to multiple nuclear DNA markers. In contrast to the nuclear genome, due to the uniparental (maternal) mode of inheritance, no individual has unique mtDNA. We investigated the possibility of increasing the discrimination power by using pyrosequencing technology to analyse parts of the coding region in addition to HVI/HVII (Paper III). Furthermore, the addition of coding mtDNA information was evaluated by comparing several recently published mtDNA coding region assays (Paper IV). Mixtures of DNA are common in forensic genetics due to contribution of DNA from several individuals, contamination or heteroplasmy. To resolve mixtures we have developed a pyrosequencing-based assay for the accurate quantification of the mtDNA mixture components (Paper V). In conclusion, this thesis describes several assays that are valuable in forensic genetics for DNA quantification, improved mtDNA analysis, and mtDNA mixture interpretation.