Cancer incidence, mortality, and pregnancy outcome among women treated for cervical intraepithelial neoplasia

Cervical cancer develops through precursor lesions, i.e. cervical intraepithelialneoplasms (CIN). These can be detected and treated before progression to invasive cancer. The major risk factor for developing cervical cancer or CIN is persistent or recurrent infection with high-risk human papilloma virus (hrHPV). Other associated risk factors include low socioeconomic status, smoking, sexually transmitted infections, and high number of sexual partners, and these risk factors can predispose to some other cancers, excess mortality, and reproductive health complications as well. The aim was to study long-term cancer incidence, mortality, and reproductive health outcomes among women treated for CIN. Based on the results, we could evaluate the efficacy and safety of CIN treatment practices and estimate the role of the risk factors of CIN patients for cancer incidence, mortality, and reproductive health. We collected a cohort of 7 599 women treated for CIN at Helsinki University Central Hospital from 1974 to 2001. Information about their cancer incidence, cause of death, birth of children and other reproductive endpoints, and socio-economic status were gathered through registerlinkages to the Finnish Cancer Registry, Finnish Population Registry, and Statistics Finland. Depending on the endpoints in question, the women treated were compared to the general population, to themselves, or to an age- and municipality-matched reference cohort. Cervical cancer incidence was increased after treatment of CIN for at least 20 years, regardless of the grade of histology at treatment. Compared to all of the colposcopically guided methods, cold knife conization (CKC) was the least effective method of treatment in terms of later CIN 3 or cervical cancer incidence. In addition to cervical cancer, incidence of other HPV-related anogenital cancers was increased among those treated, as was the incidence of lung cancer and other smoking-related cancers. Mortality from cervical cancer among the women treated was not statistically significantly elevated, and after adjustment for socio-economic status, the hazard ratio (HR) was 1.0. In fact, the excess mortality among those treated was mainly due to increased mortality from other cancers, especially from lung cancer. In terms of post-treatment fertility, the CIN treatments seem to be safe: The women had more deliveries, and their incidence of pregnancy was similar before and after treatment. Incidence of extra-uterine pregnancies and induced abortions was elevated among the treated both before and after treatment. Thus this elevation did not occur because they were treated rather to a great extent was due to the other known risk factors these women had in excess, i.e. sexually transmitted infections. The purpose of any cancer preventive activity is to reduce cancer incidence and mortality. In Finland, cervical cancer is a rare disease and death from it even rarer, mostly due to the effective screening program. Despite this, the women treated are at increased risk for cancer; not just for cervical cancer. They must be followed up carefully and for a long period of time; general health education, especially cessation of smoking, is crucial in the management process, as well as interventions towards proper use of birth control such as condoms.

Peripheral facial palsy : Grading, Etiology, and Melkersson-Rosenthal Syndrome

The objective of this study was to assess the utility of two subjective facial grading systems, to evaluate the etiologic role of human herpesviruses in peripheral facial palsy (FP), and to explore characteristics of Melkersson-Rosenthal syndrome (MRS). Intrarater repeatability and interrater agreement were assessed for Sunnybrook (SFGS) and House-Brackmann facial grading systems (H-B FGS). Eight video-recorded FP patients were graded in two sittings by 26 doctors. Repeatability for SFGS was from good to excellent and agreement between doctors from moderate to excellent by intraclass correlation coefficient and coefficient of repeatability. For H-B FGS, repeatability was from fair to good and agreement from poor to fair by agreement percentage and kappa coefficients. Because SFGS was at least as good in repeatability as H-B FGS and showed more reliable results in agreement between doctors, we encourage the use of SFGS over H-B FGS. Etiologic role of human herpesviruses in peripheral FP was studied by searching DNA of herpes simplex virus (HSV) -1 and -2, varicella-zoster virus (VZV), human herpesvirus (HHV) -6A, -6B, and -7, Epstein-Barr virus (EBV), and cytomegalovirus (CMV) by PCR/microarray methods in cerebrospinal fluid (CSF) of 33 peripheral FP patients and 36 controls. Three patients and five controls had HHV-6 or -7 DNA in CSF. No DNA of HSV-1 or -2, VZV, EBV, or CMV was found. Detecting HHV-7 and dual HHV-6A and -6B DNA in CSF of FP patients is intriguing, but does not allow etiologic conclusions as such. These DNA findings in association with FP and the other diseases that they accompanied require further exploration. MRS is classically defined as a triad of recurrent labial or oro-facial edema, recurrent peripheral FP, and plicated tongue. All three signs are present in the minority of patients. Edema-dominated forms are more common in the literature, while MRS with FP has received little attention. The etiology and true incidence of MRS are unknown. Characteristics of MRS were evaluated at the Departments of Otorhinolaryngology and Dermatology focusing on patients with FP. There were 35 MRS patients, 20 with FP and they were mailed a questionnaire (17 answered) and were clinically examined (14 patients). At the Department of Otorhinolaryngology, every MRS patient had FP and half had the triad form of MRS. Two patients, whose tissue biopsies were taken during an acute edema episode, revealed nonnecrotizing granulomatous findings typical for MRS, the other without persisting edema and with symptoms for less than a year. A peripheral blood DNA was searched for gene mutations leading to UNC-93B protein deficiency predisposing to HSV-1 infections; no gene mutations were found. Edema in most MRS FP patients did not dominate the clinical picture, and no progression of the disease was observed, contrary to existing knowledge. At the Department of Dermatology, two patients had triad MRS and 15 had monosymptomatic granulomatous cheilitis with frequent or persistent edema and typical MRS tissue histology. The clinical picture of MRS varied according to the department where the patient was treated. More studies from otorhinolaryngology departments and on patients with FP would clarify the actual incidence and clinical picture of the syndrome.

Healthcare-Associated Infections in Pediatrics

Healthcare-associated infections (HAIs) are known to increase the risk for patient morbidity and mortality in different healthcare settings and thereby to cause additional costs. HAIs typically affect patients with severe underlying conditions. HAIs are prevalent also among pediatric patients, but the distribution of the types of infection and the causative agents differ from those detected in adults. The aim of this study was to obtain information on pediatric HAIs in Finland through an assessment of the surveillance of bloodstream infections (BSIs), through two outbreak investigations in a neonatal intensive care unit (NICU), and through a study of postoperative HAIs after open-heart surgery. The studies were carried out at the Hospital for Children and Adolescents of Helsinki University Central Hospital. Epidemiological features of pediatric BSIs were assessed. For the outbreak investigations, case definitions were set and data collected from microbiological and clinical records. The antimicrobial susceptibilities of the Serratia marcescens and the Candida parapsilosis isolates were determined and they were genotyped. Patient charts were reviewed for the case-control and cohort studies during the outbreak investigations, as well as for the patients who acquired surgical site infections (SSIs) after having undergone open-heart surgery. Also a prospective postdischarge study was conducted to detect postoperative HAIs in these patients. During 1999-2006, the overall annual BSI rate was 1.6/1,000 patient days (range by year, 1.2–2.1). High rates (average, 4.9 and 3.2 BSIs/1,000 patient days) were detected in hematology and neonatology units. Coagulase-negative staphylococci were the most common pathogens both hospital-wide and in each patient group. The overall mortality was 5%. The genotyping of the 15 S. marcescens isolates revealed three independent clusters. All of the 26 C. parapsilosis isolates studied proved to be indistinguishable. The NICU was overcrowded during the S. marcescens clusters. A negative correlation between C. parapsilosis BSIs and fluconazole use in the NICU was detected, and the isolates derived from a single initially susceptible strain became less susceptible to fluconazole over time. Eighty postoperative HAIs, including all severe infections, were detected during hospitalization after open-heart surgery; 34% of those HAIs were SSIs and 25% were BSIs. The postdischarge study found 65 infections that were likely to be associated with hospitalization. The majority (89%) of them were viral respiratory or gastrointestinal infections, and these often led to rehospitalizations. The annual hospital-wide BSI rates were stable, and the significant variation detected in some units could not be seen in overall rates. Further studies with data adequately adjusted for risk factors are needed to assess BSI rates in the patient groups with the highest rates (hematology, neonatology). The outbreak investigations showed that horizontal transmission was common in the NICU. Overcrowding and lapses in hand hygiene probably contributed to the spreading of the pathogens. Following long-term use of fluconazole in the NICU, resistance to fluconazole developed in C. parapsilosis. Almost one-fourth of the patients who underwent open-heart surgery acquired at least one HAI. All severe HAIs were detected during hospitalization. The postdischarge study found numerous viral infections, which often caused rehospitalization.

Resistance responses blocking systemic movement of Potato virus A in potato

Peruna kestää A-virusta estämällä sen leviämistä Peruna on maissin ohella maailman kolmanneksi tärkein ravintokasvi vehnän ja riisin jälkeen. Perunaa lisätään kasvullisesti mukuloita istuttamalla, jolloin virukset siirtyvät sairaiden siemenmukuloiden välityksellä kasvukaudesta toiseen. Virustauteja voi torjua ainoastaan terveen siemenperunan ja kestävien lajikkeiden avulla. Kestävyys perustuu usein siihen, että kasvi estää viruksen leviämisen tartuntakohdasta välttyäkseen virustaudilta. Tässä työssä tutkittiin kolmea perunan A-viruksen (PVA) liikkumista estävää kestävyysmekanismia perunassa. Lisäksi työn kokeelliseen osaan oleellisesti kuuluvaa virustartutusta varten kehitettiin uusi paranneltu versio geenipyssystä. Tämä itse rakennettu laite optimoitiin PVA:n tartuttamiseen mahdollisimman helposti ja pienin käyttökustannuksin. Tutkimuksen kohteena olleessa perunan risteytysjälkeläistössä oli PVA:ta kestäviä kasveja (ryhmä nnr), jotka estivät viruksen liikkumisen aiheuttamatta oireita tartutuskohdassa, sekä kasveja, joissa PVA aiheutti kuolioläikkinä näkyvän yliherkkyysvasteen (ryhmä HR). Molemmissa kestävyystyypeissä virus pystyi monistumaan ja leviämään solusta soluun paikallisesti, mutta liikkuminen muihin kasvinosiin nilan kautta estyi. Ryhmän nnr kasveissa PVA-tartunta ei aiheuttanut tilastollisesti merkitsevää muutosta useimpien geenien ilmenemiseen tartuntakohdassa. Ainoastaan geeniperhe, joka ilmentää tiettyä proteinaasi-inhibiittoria (PI), reagoi PVA:han 24 tuntia tartutuksesta. Kun tämän PVA:han reagoivan geeniperheen jäsenet hiljennettiin nnr- perunalinjoissa, ne muuttuivat alttiiksi PVA:lle ja virus levisi tartuntakohdasta muihin kasvinosiin. Tulos osoittaa, että PI on viruskestävyystekijä. Lisäksi muut tutkimuksessa saadut tulokset tukevat mahdollisuutta, että PI estää PVA:n P1-proteinaasin toimintaa. HR-linjoissa todettiin erilaisiin puolustusvasteisiin liittyvien PR-geenien aktivoitumista PVA-tartunnan seurauksena, mutta myös ilman sitä kasvien kasvettua mullassa noin neljä viikkoa. Sen sijaan solukkoviljelyssä tai vasta kaksi viikkoa mullassa kasvaneissa kasveissa vastaavaa ei vielä todettu. Tulos viittaa siihen, että HR-perunat reagoivat herkemmin ympäristöön ja/tai kasvin kehitysasteeseen laukaisten puolustusvasteita, jotka saattavat parantaa kestävyyttä taudinaiheuttajia vastaan. Kolmas tutkittu kestävyystyyppi havaittiin Pito-perunalajikkeessa. Se muistutti nnr-kestävyyttä siten, että myös siinä viruksen liikkuminen nilassa muihin kasvinosiin estyi. PVA:n todettiin pysähtyvän vasta lehtiruodin tyvelle muodostuvaan irtoamisvyöhykkeeseen, mitä havainnollistettiin käyttämällä muunnettua PVA-rotua, joka tuotti UV-valossa fluoresoivaa vihreää valoa. Tulos viittaa siihen, että virus ei pääse kulkemaan vyöhykkeeseen kuuluvan suojaavan kerroksen läpi, jollei sillä ole pääsyä nilaan. Tällainen kestävyys on tarpeen, jotta virus ei voi korvata nilakuljetusta solusta soluun leviämisellä. Tulokset tuovat uusia näkökulmia kasvien viruskestävyyteen ja auttavat selittämään viruksen nilakuljetuksen estymistä sekä solusta soluun leviämisen pysähtymistä kestävissä kasveissa.

Simplified Treatment of Childhood Acute Bone and Joint Infections

Acute childhood osteomyelitis (OM), septic arthritis (SA), and their combination osteomyelitis with adjacent septic arthritis (OM+SA), are treated with long courses of antimicrobials and immediate surgery. We conducted a prospective multi-center randomized trial among Finnish children at age 3 months to 15 years in 1983-2005. According to the two-by-two factorial study design, children with OM or OM+SA received 20 or 30 days of antimicrobials, whereas those with SA were treated for 10 or 30 days. In addition, the whole series was randomized to be treated with clindamycin or a first-generation cephalosporin. Cases were included only if the causative agent was isolated. The treatment was instituted intravenously, but only for the first 2-4 days. Percutaneous aspiration was done to obtain a representative sample for bacteriology, but all other surgical intervention was kept at a minimum. A total of 265 patients fulfilled our strict inclusion criteria and were analyzed; 106 children had OM, 134 SA, and 25 OM+SA. In the OM group, one child in the long and one child in the short-term treatment group developed sequelae. One child with SA twice developed a late re-infection of the same joint, but the causative agents differed. Regarding surgery, diagnostic arthrocentesis or corticotomy was the only surgical procedure performed in most cases. Routine arthrotomy was not required even in hip arthritis. Serum C-reactive protein (CRP) proved to be a reliable laboratory index in the diagnosis and monitoring of osteoarticular infections. The recovery rate was similar regardless of whether clindamycin or a first-generation cephalosporin was used. We conclude that a course of 20 days of these well-absorbing antimicrobials is sufficient for OM or OM+SA, and 10 days for SA in most cases beyond the neonatal age. A short intravenous phase of only 2-5 days often suffices. CRP gives valuable information in monitoring the course of illness. Besides diagnostic aspiration, surgery should be reserved for selected cases.

Human parvovirus infections during pregnancy : Special reference to the child-care employees

Human parvovirus B19 (B19V) is known to cause anemia, hydrops fetalis, and fetal death especially during the first half of pregnancy. Women who are in occupational contact with young children are at increased risk of B19V infection. The role of the recently discovered human parvovirus, human bocavirus (HBoV), in reproduction is unknown. The aim of this research project was to establish a scientific basis for assessing the work safety of pregnant women and for issuing special maternity leave regulations during B19V epidemics in Finland. The impact of HBoV infection on the pregnant woman and her fetus was also defined. B19V DNA was found in 0.8% of the miscarriages and in 2.4% of the intrauterine fetal death (IUFD; fetal death after completed 22 gestational weeks). All control fetuses (from induced abortions) were B19V-DNA negative. The findings on hydropic B19V DNA-positive IUFDs with evidence of acute or recent maternal B19V infection are in line with those of previous Swedish studies. However, the high prevalence of B19V-related nonhydropic IUFDs noted in the Swedish studies was mostly without evidence of maternal B19V infection and was not found during the third trimester. HBoV was not associated with miscarriages or IUFDs. Almost all of the studied pregnant women were HboV-IgG positive, and thus most probably immune to HBoV. All preterm births, perinatal deaths, smallness for gestational age (SGA) and congenital anomaly were recorded among the infants of child-care employees in a nationwide register-based cohort study over a period of 14 years. Little or no differences in the results were found between the infants of the child-care employees and those of the comparison group. The annual B19V seroconversion rate was over two-fold among the child-care employees, compared to the women in the comparison group. The seropositivity of the child-care employees increased with age, and years from qualification/joining the trade union. In general, the child-care employees are not at increased risk for adverse pregnancy outcome. However, at the population level, the risk of rare events, such as adverse pregnancy outcomes attributed to infections, could not be determined. According to previous studies, seronegative women had a 5 10% excess risk of losing the fetus during the first half of their pregnancy, but thereafter the risk was very low. Therefore, an over two-fold increased risk of B19V infection among child-care employees is considerable, and should be taken into account in the assessment of the occupational safety of pregnant women, especially during the first half of their pregnancy.

The determinants of sexually transmitted infections among reproductive age women in St. Petersburg, Estonia and Finland

OBJECTIVES: Sexually transmitted infections' (STIs) rate vary in St. Petersburg, Estonia and Finland; the aim was to compare the determinants of self-reported sexually transmitted infections in these areas. METHODS: Data from four population-based questionnaire surveys were used (Finland in 1992 and 1999; St. Petersburg in 2003; Estonia in 2004). With the exception of the 1992 Finnish survey (interview) all were postal surveys, with 1,070 respondents in Finland (78 and 52% response rates), 1,147 (68%) in St. Petersburg, and 5,190 (54%) in Estonia. RESULTS: Risky sexual behaviours were equally common in the three areas and the determinants were the same. Women with an STIs history more often had had their first sexual intercourse when aged under 18, had not used condom during first intercourse, had a high number of lifetime or previous year sexual partners. However, marital status and education were not similar determinants. Cohabiting and well-educated women in Finland were more likely to have STIs while in other areas the associations found were not statistically significant. CONCLUSIONS: Risky behaviour predicts STIs, but does not explain the varying rates of STIs between areas.

Perunan Y-viruksen testaaminen korkeassa happi-hiilidioksidipitoisuudessa idätetyistä perunoista

Perunalla (Solanum tuberosum L.) tällä hetkellä maailmanlaajuisesti eniten sato- ja laatutappioita aiheuttaa perunan Y-virus (PVY). Vaikka pelkän Y-viruksen aiheuttamaa satotappiota on vaikea mitata, on sen arvioitu olevan 20-80 %. Viruksen tärkein leviämistapa on viroottinen siemenperuna. Korkealaatuinen siemenperuna on edellytys ruoka-, ruokateollisuus- ja tärkkelysperunan tuotannolle. Kasvuston silmämääräinen tarkastelu aliarvioi yleensä Y-viruksen esiintyvyyttä. Laboratoriotestauksen avulla saadaan tarkempi tieto pellolta korjatun sadon saastunta-asteesta. Ongelmana Y-viruksen testaamisessa on, että sitä ei havaita dormanssissa olevista perunoista otetuista näytteistä yhtä luotettavasti kuin jo dormanssin ohittaneista perunoista testattaessa. Erilaisia menetelmiä kemikaaleista (Rindite, bromietaani) kasvihormoneihin (mm. gibberelliinihappo) ja varastointiolosuhteiden muutoksiin (kylmä- ja lämpökäsittely) on kokeiltu perunan dormanssin purkamiseen, mutta tulokset ovat olleet vaihtelevia. Tässä tutkielmassa perunan dormanssin purkamiseen käytettiin happi-hiilidioksidikäsittelyä (O2 40 % ja CO2 20 %) eripituisina käsittelyaikoina. Tarkoituksena oli selvittää, vaikuttaako käsittely perunan itämiseen ja dormanssin luontaista aikaisempaan purkautumiseen tai Y-viruksen havaitsemiseen. Lisäksi haluttiin selvittää, voiko Y-viruksen määrittämisen ELISA-testillä (Enzyme Linked Immunosorbent Assay) tehdä yhtä luotettavasti myös muista kasvinosista (mukula, itu), kuin tällä hetkellä yleisesti käytetystä perunan lehdestä. Idätyskäsittelyn vaikutuksista dormanssin purkautumiseen saatiin vaihtelevia, eikä kovinkaan yleistettäviä tuloksia. Käsittelyn ei myöskään havaittu vaikuttavan PYY-viroottisuuden havaitsemiseen eri näytemateriaaleilla testattaessa. Kun eri kasvinosien toimivuutta testissä vertailtiin, mukulamateriaalin todettiin aliarvioivan PVY-viroottisuutta kaikissa kokeissa. Myös itumateriaali aliarvioi pääsääntöisesti PVY-viroottisuutta ELISA:lla tehdyissä määrityksissä. Luotettavin testimateriaali oli perunan lehti.

Molecular Characterization of Viruses Causing the Cassava Brown Streak Disease Epidemic in Eastern Africa

Cassava brown streak disease (CBSD) was described for the first time in Tanganyika (now Tanzania) about seven decades ago. Tanganyika (now Tanzania) about seven decades ago. It was endemic in the lowland areas of East Africa and inland parts of Malawi and caused by Cassava brown streak virus (CBSV; genus Ipomovirus; Potyviridae). However, in 1990s CBSD was observed at high altitude areas in Uganda. The causes for spread to new locations were not known.The present work was thus initiated to generate information on genetic variability, clarify the taxonomy of the virus or viruses associated with CBSD in Eastern Africa as well as to understand the evolutionary forces acting on their genes. It also sought to develop a molecular based diagnostic tool for detection of CBSD-associated virus isolates. Comparison of the CP-encoding sequences of CBSD-associated virus isolates collected from Uganda and north-western Tanzania in 2007 and the partial sequences available in Genbank revealed occurrence of two genetically distinct groups of isolates. Two isolates were selected to represent the two groups. The complete genomes of isolates MLB3 (TZ:Mlb3:07) and Kor6 (TZ:Kor6:08) obtained from North-Western (Kagera) and North-Eastern (Tanga) Tanzania, respectively, were sequenced. The genomes were 9069 and 8995 nucleotides (nt), respectively. They translated into polyproteins that were predicted to yield ten mature proteins after cleavage. Nine proteins were typical in the family Potyviridae, namely P1, P3, 6K1, CI, 6K2, VPg, NIa-Pro, NIb and CP, but the viruses did not contain HC-Pro. Interestingly, genomes of both isolates contained a Maf/HAM1-like sequence (HAM1h; 678 nucleotides, 25 kDa) recombined between the NIb and CP domains in the 3’-proximal part of the genomes. HAM1h was also identified in Euphorbia ringspot virus (EuRSV) whose sequence was in GenBank. The HAM1 gene is widely spread in both prokaryotes and eukaryotes. In yeast (Saccharomyces cerevisiae) it is known to be a nucleoside triphosphate (NTP) pyrophosphatase. Novel information was obtained on the structural variation at the N-termini of polyproteins of viruses in the genus Ipomovirus. Cucumber vein yellowing virus (CVYV) and Squash vein yellowing virus (SqVYV) contain a duplicated P1 (P1a and P1b) but lack the HC-Pro. On the other hand, Sweet potato mild mottle virus (SPMMV), has a single but large P1 and has HC-Pro. Both virus isolates (TZ:Mlb3:07 & TZ:Kor6:08) characterized in this study contained a single P1 and lacked the HC-Pro which indicates unique evolution in the family Potyviridae. Comparison of 12 complete genomes of CBSD-associated viruses which included two genomes characterized in this study, revealed genetic identity of 69.0–70.3% (nt) and amino acid (aa) identities of 73.6–74.4% at polyprotein level. Comparison was also made among 68 complete CP sequences, which indicated 69.0-70.3 and 73.6-74.4 % identity at nt and aa levels, respectively. The genetic variation was large enough for dermacation of CBSD-associated virus isolates into two distinct species. The name CBSV was retained for isolates that were related to CBSV isolates available in database whereas the new virus described for the first time in this study was named Ugandan cassava brown streak virus (UCBSV) by the International Committee on Virus Taxonomy (ICTV). The isolates TZ:Mlb3:07 and TZ:Kor6:08 belong to UCBSV and CBSV, respectively. The isolates of CBSV and UCBSV were 79.3-95.5% and 86.3-99.3 % identitical at nt level, respectively, suggesting more variation amongst CBSV isolates. The main sources of variation in plant viruses are mutations and recombination. Signals for recombination events were detected in 50% of isolates of each virus. Recombination events were detected in coding and non-coding (3’-UTR) sequences except in the 5’UTR and P3. There was no evidence for recombination between isolates of CBSV and UCBSV. The non-synonomous (dN) to synonomous (dS) nucleotide substitution ratio (ω) for the HAM1h and CP domains of both viruses were ≤ 0.184 suggesting that most sites of these proteins were evolving under strong purifying selection. However, there were individual amino acid sites that were submitted to adaptive evolution. For instance, adaptive evolution was detected in the HAM1h of UCBSV (n=15) where 12 aa sites were under positive selection (P< 0.05) but not in CBSV (n=12). The CP of CBSV (n=23) contained 12 aa sites (p<0.01) while only 5 aa sites in the CP gene of UCBSV were predicted to be submitted to positive selection pressure (p<0.01). The advantages offered by the aa sites under positive selection could not be established but occurrence of such sites in the terminal ends of UCBSV-HAMIh, for example, was interpreted as a requirement for proteolysis during polyprotein processing. Two different primer pairs that simultaneously detect UCBSV and CBSV isolates were developed in this study. They were used successfully to study distribution of CBSV, UCBSV and their mixed infections in Tanzania and Uganda. It was established that the two viruses co-infect cassava and that incidences of co-infection could be as high as 50% around Lake Victoria on the Tanzanian side. Furthermore, it was revealed for the first time that both UCBSV and CBSV were widely distributed in Eastern Africa. The primer pair was also used to confirm infection in a close relative of cassava, Manihot glaziovii (Müller Arg.) with CBSV. DNA barcoding of M. glaziovii was done by sequencing the matK gene. Two out of seven M. glaziovii from the coastal areas of Korogwe and Kibaha in north eastern Tanzania were shown to be infected by CBSV but not UCBSV isolates. Detection in M. glaziovii has an implication in control and management of CBSD as it is likely to serve as virus reservoir. This study has contributed to the understanding of evolution of CBSV and UCBSV, which cause CBSD epidemic in Eastern Africa. The detection tools developed in this work will be useful in plant breeding, verification of the phytosanitary status of materials in regional and international movement of germplasm, and in all diagnostic activities related to management of CBSD. Whereas there are still many issues to be resolved such as the function and biological significance of HAM1h and its origin, this work has laid a foundation upon which the studies on these aspects can be based.

Development of tissue culture and virus-induced gene silencing for Calendula officinalis

Calendula officinalis is grown widely as an ornamental plant across Europe. It belongs to the large. Asteraceae family. In this study, the aim was to explore the possibilities to use Calendula officinalis as a new model organism for flower development and secondary mechanism studies in Asteraceae. Tissue culture of Calendula officinalis was established using nine different cultivars. Murashige & Skoog (MS) medium with four different combinations of plant growth regulators were tested. Of all these combinations, the medium containing 1mg/l BAP, 0.1 mg/l IAA, and 1mg/l Zeatin achieved highest frequency of adventitious shoot regeneration from hypocotyl and cotyledon explants. Virus-induced gene silencing is a recent developed genetic tool for charactering the gene functions in plants, and extends the range of host plants that are not accessible for Agrobacterium transformation. Here, tobacco rattle virus (TRV)-based VIGS technique was tested in calendula (cv. Single Orange). We used TRV carrying Gerbera hybrid phytoene desaturase (PDS) gene fragment to induce PDS silencing in calendula. Vacuum infiltration and syringe infiltration methods both resulted in photo-bleaching phenotypes in leaves, bracts and petals. Loss-of-function phenotypes occurred on calendula 13 days post-infiltration. In conclusion, the data indicates that calendula explants can be regenerated through tissue culture which is a prerequisite for development of stable transformation methods. However, further optimization is still needed to improve the frequency. In addition, VIGS was applied to silence PDS marker gene expression indicating that this method has potential for gene functional studies in future.

Basic and translational studies on species B adenoviruses

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.