Cervical cancer is preceded by a spectrum of abnormalities in the cervical epithelium. Research supports an etiological role for certain types of human papillomaviruses (HPVs) in cervical pathology. More than 70 HPV genotypes have been characterized based on complete genome sequences and of these, about half infect the genital tract. Genital HPVs are further classified as either "high risk" or "low risk" types based on their association with cervical cancer. The demonstration that there is a relationship between HPV infection and cervical cancer has been dependent on a number of viral nucleic acid-based detection systems such as Southern blot and polymerase chain reaction. However, the lack of methods which discriminate between a specific HPV type and a large number of related HPV genotypes has made studies of disease association difficult. In the first part of this study, a recently developed Amplicor HPV Genotyping Kit was evaluated with respect to its ability to define HPV infection status. The L1 region was directly sequenced from the PCR product in 16 clinical samples to determine which genotype(s) was/were present. Sequencing data from one sample suggested a mixed infection and therefore the PCR product was cloned and sequenced to see if more than one genotype was present. Fifteen out of the sixteen samples sequenced were HPV genotypes which are not represented on the Amplicor Genotyping Kit test strips. The samples are rare HPV types (HPV 61, 62, CP6108 and CP8304). The second part of this study examined the issue of persistence and in particular, I have considered the issue of an appropriate definition of persistence. A number of patients who had evidence of HPV 16 or HPV 18, 6 and 66 were investigated using samples obtained on at least two occasions. Molecular variants of either the LCR gene for HPV 16 or the L1 gene for the other HPV types were studied. No differences in LCR or L1 gene sequence in sequential same patient samples were observed. Two HPV 16 LCR alleles were seen for six patients of which 8/10 showed one nucleotide change at base 7518 and 2/10 were identical to the prototype. Based on a compilation of published studies on HPV 16 variants and subtypes for the long control region, the number of HPV 16 LCR alleles, worldwide, was determined. One HPV 66 and two HPV 18 and HPV 6 L1 alleles were observed. When examining persistence, one must consider the frequency of specific variants (alleles) in the study population. A common variant detected over time could either be a persistent infection or a reinfection with the same viral variant. / Thesis / Master of Science (MS)

Background Arboviral diseases are one of the most common causes of acute febrile illness (AFI) and a significant health problem in South America. In Peru, laboratory etiologic identification of these infections occurs in less than 50% of cases, leading to underdiagnoses of important emerging arboviruses. Aim To assess the prevalence of the Dengue (DENV), Oropouche (OROV), Chikungunya (CHIKV), Mayaro (MAYV) and Zika (ZIKV) viruses in patients with acute febrile illness from Puerto Maldonado (Peru). Methodology Serum samples were obtained from patients with AFI during January 2016 to March 2016. A total of 139 specimens were analyzed for the presence of DENV, OROV, CHIKV, MAYV, and ZIKV using polymerase chain reaction (PCR). Results CHIKV in 9.4% and OROV in 8.6% were the most prevalent arboviruses, followed by DENV and ZIKV, with a prevalence of 6.5% and 5%, respectively. Among all patients, the most common symptoms accompanying fever were headaches 79.9%, muscle pain 65.5% and joint pain 63.3%. Conclusions During this short 3-month period, 4 arboviruses were detected by PCR, CHIKV and OROV being the most common arboviruses in Puerto Maldonado (Peru). Thus, it is crucial to include OROV detection in the national health surveillance. Furthermore, the etiologic clinical diagnosis of arboviral infections is not possible due to the low specificity of symptoms; therefore an increase of cases confirmed by molecular diagnostic methods will enhance arboviral surveillance in Peru.

The overall objective of this study was to test the hypothesis that insects can vector F. tumidum conidia to infect gorse plants with the aim of developing an alternative approach to mycoherbicide delivery to control weeds. Four potential insect species (Apion ulicis, Cydia ulicetana, Epiphyas postvittana and Sericothrips staphylinus) were assessed for their ability to vector F. tumidum conidia. To achieve this, the external microflora (bacteria and fungi) and the size and location of fungal spores on the cuticle of these insect species were determined. In addition, the ability of the insects to pick up and deposit F. tumidum conidia on agar was studied. Based on the results from these experiments, E. postvittana was selected for more detailed experiments to determine transmission of F. tumidum to infect potted gorse plants. The factors promoting pathogenicity of F. tumidum against gorse and the pathogen loading required to infect and kill the weed were also determined. The external microflora of the four insect species were recovered by washing and plating techniques and identified by morphology and polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) and sequencing of internally transcribed spacer (ITS) and 16S rDNA. A culture-independent technique (direct PCR) was also used to assess fungal diversity by direct amplification of ITS sequences from the washings of the insects. All insect species carried Alternaria, Cladosporium, Nectria, Penicillium, Phoma, Pseudozyma spp. and entomopathogens. Ninety four per cent of the 178 cloned amplicons had ITS sequences similarity to Nectria mauritiicola. E. postvittana carried the largest fungal spores (mean surface area of 125.9 ìm2) and the most fungal CFU/insect. About 70% of the fungi isolated from the insects were also present on the host plant (gorse) and the understorey grass. The mean size of fungal spores recovered from the insect species correlated strongly with their body length (R² = 85%). Methylobacterium aquaticum and Pseudomonas lutea were common on all four insect species. Pseudomonas fluorescens was the most abundant bacterial species. In the pathogenicity trials, the effectiveness of F. tumidum in reducing root and shoot biomass of 16 and 8 wk old gorse plants was significantly increased with wounding of the plants. Older plants (32 wk old) which were wounded and inoculated were significantly shorter, more infected and developed more tip dieback (80%) than plants which were not wounded (32%). This indicates that damage caused by phytophagous insect species present on gorse through feeding and oviposition may enhance infection by F. tumidum. Wounding may release nutrients (e.g. Mg and Zn) essential for conidia germination and germ tube elongation and also provide easier access for germ tube penetration. Conidial germination and germ tube length were increased by 50 and 877%, respectively when incubated in 0.2% of gorse extract solution for 24 h compared with incubation in water. Inoculum suspensions amended with 0.2% of gorse extract caused more infection and significantly reduced biomass production of 24 wk old gorse plants than suspensions without gorse extract. A minimum number of about 900 viable conidia/infection site of F. tumidum were required to infect gorse leaves. However, incorporation of amendments (which can injure the leaf cuticle) or provision of nutrients (i.e. gorse extract or glucose) in the formulation might decrease the number of conidia required for lesion formation. Scanning electron micrographs showed that germ tube penetration of gorse tissue was limited to open stomata which partly explain the large number of conidia required for infection. The flowers and leaves were more susceptible to F. tumidum infection than the spines, stems and pods. An experiment to determine the number of infection sites required to cause plant mortality showed that the entire plant needs to be inoculated in order for the pathogen to kill 10 wk old plants as F. tumidum is a non systemic pathogen. The number of infection sites correlated strongly with disease severity (R² = 99.3%). At least 50% of the plant was required to be inoculated to cause a significant reduction in shoot dry weight. F. tumidum, applied as soil inoculant using inoculated wheat grains in three separate experiments, significantly suppressed gorse seedling emergence and biomass production. In experiments to determine the loading capacity of the insect species, E. postvittana, the largest insect species studied, carried significantly more (68) and deposited significantly more (29) F. tumidum conidia than the other species. Each E. postvittana, loaded with 5,000 conidia of F. tumidum, transmitted approximately 310 conidia onto gorse plants but this did not cause any infection or affect plant growth as determined by shoot fresh weight and shoot height. E. postvittana on its own did not cause any significant damage to gorse and did not enhance F. tumidum infection. It also failed to spread the pathogen from infected plants to the healthy ones. There was no evidence of synergism between the two agents and damage caused by the combination of both E. postvittana and F. tumidum was equivalent to that caused by F. tumidum alone. This study has shown that E. postvittana has the greatest capacity to vector F. tumidum since it naturally carried the largest and the most fungal spores (429 CFU/insect). Moreover, it naturally carried Fusarium spp. such as F. lateritium, F. tricinctum and Gibberella pulicaris (anamorph Fusarium sambucinum) and was capable of carrying and depositing most F. tumidum conidia on agar. Coupled with the availability of pheromone for attracting the male insects, E. postvittana may be a suitable insect vector for delivering F. tumidum conidia on gorse using this novel biocontrol strategy. Although it is a polyphagous insect, and may visit non-target plants, F. tumidum is a very specific pathogen of gorse, broom and a few closely related plant species. Hence, using this insect species to vector F. tumidum in a biological control programme, should not pose a significant threat to plants of economic importance. However, successful control of gorse using this "lure-load-infect" concept would depend, to a large extent on the virulence of the pathogen as insects, due to the large size of F. tumidum macroconidia, can carry only a small number of it.

The overall objective of this study was to test the hypothesis that insects can vector F. tumidum conidia to infect gorse plants with the aim of developing an alternative approach to mycoherbicide delivery to control weeds. Four potential insect species (Apion ulicis, Cydia ulicetana, Epiphyas postvittana and Sericothrips staphylinus) were assessed for their ability to vector F. tumidum conidia. To achieve this, the external microflora (bacteria and fungi) and the size and location of fungal spores on the cuticle of these insect species were determined. In addition, the ability of the insects to pick up and deposit F. tumidum conidia on agar was studied. Based on the results from these experiments, E. postvittana was selected for more detailed experiments to determine transmission of F. tumidum to infect potted gorse plants. The factors promoting pathogenicity of F. tumidum against gorse and the pathogen loading required to infect and kill the weed were also determined. The external microflora of the four insect species were recovered by washing and plating techniques and identified by morphology and polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) and sequencing of internally transcribed spacer (ITS) and 16S rDNA. A culture-independent technique (direct PCR) was also used to assess fungal diversity by direct amplification of ITS sequences from the washings of the insects. All insect species carried Alternaria, Cladosporium, Nectria, Penicillium, Phoma, Pseudozyma spp. and entomopathogens. Ninety four per cent of the 178 cloned amplicons had ITS sequences similarity to Nectria mauritiicola. E. postvittana carried the largest fungal spores (mean surface area of 125.9 µm²) and the most fungal CFU/insect. About 70% of the fungi isolated from the insects were also present on the host plant (gorse) and the understorey grass. The mean size of fungal spores recovered from the insect species correlated strongly with their body length (R² = 85%). Methylobacterium aquaticum and Pseudomonas lutea were common on all four insect species. Pseudomonas fluorescens was the most abundant bacterial species. In the pathogenicity trials, the effectiveness of F. tumidum in reducing root and shoot biomass of 16 and 8 wk old gorse plants was significantly increased with wounding of the plants. Older plants (32 wk old) which were wounded and inoculated were significantly shorter, more infected and developed more tip dieback (80%) than plants which were not wounded (32%). This indicates that damage caused by phytophagous insect species present on gorse through feeding and oviposition may enhance infection by F. tumidum. Wounding may release nutrients (e.g. Mg and Zn) essential for conidia germination and germ tube elongation and also provide easier access for germ tube penetration. Conidial germination and germ tube length were increased by 50 and 877%, respectively when incubated in 0.2% of gorse extract solution for 24 h compared with incubation in water. Inoculum suspensions amended with 0.2% of gorse extract caused more infection and significantly reduced biomass production of 24 wk old gorse plants than suspensions without gorse extract. A minimum number of about 900 viable conidia/infection site of F. tumidum were required to infect gorse leaves. However, incorporation of amendments (which can injure the leaf cuticle) or provision of nutrients (i.e. gorse extract or glucose) in the formulation might decrease the number of conidia required for lesion formation. Scanning electron micrographs showed that germ tube penetration of gorse tissue was limited to open stomata which partly explain the large number of conidia required for infection. The flowers and leaves were more susceptible to F. tumidum infection than the spines, stems and pods. An experiment to determine the number of infection sites required to cause plant mortality showed that the entire plant needs to be inoculated in order for the pathogen to kill 10 wk old plants as F. tumidum is a non systemic pathogen. The number of infection sites correlated strongly with disease severity (R² = 99.3%). At least 50% of the plant was required to be inoculated to cause a significant reduction in shoot dry weight. F. tumidum, applied as soil inoculant using inoculated wheat grains in three separate experiments, significantly suppressed gorse seedling emergence and biomass production. In experiments to determine the loading capacity of the insect species, E. postvittana, the largest insect species studied, carried significantly more (68) and deposited significantly more (29) F. tumidum conidia than the other species. Each E. postvittana, loaded with 5,000 conidia of F. tumidum, transmitted approximately 310 conidia onto gorse plants but this did not cause any infection or affect plant growth as determined by shoot fresh weight and shoot height. E. postvittana on its own did not cause any significant damage to gorse and did not enhance F. tumidum infection. It also failed to spread the pathogen from infected plants to the healthy ones. There was no evidence of synergism between the two agents and damage caused by the combination of both E. postvittana and F. tumidum was equivalent to that caused by F. tumidum alone. This study has shown that E. postvittana has the greatest capacity to vector F. tumidum since it naturally carried the largest and the most fungal spores (429 CFU/insect). Moreover, it naturally carried Fusarium spp. such as F. lateritium, F. tricinctum and Gibberella pulicaris (anamorph Fusarium sambucinum) and was capable of carrying and depositing most F. tumidum conidia on agar. Coupled with the availability of pheromone for attracting the male insects, E. postvittana may be a suitable insect vector for delivering F. tumidum conidia on gorse using this novel biocontrol strategy. Although it is a polyphagous insect, and may visit non-target plants, F. tumidum is a very specific pathogen of gorse, broom and a few closely related plant species. Hence, using this insect species to vector F. tumidum in a biological control programme, should not pose a significant threat to plants of economic importance. However, successful control of gorse using this "lure-load-infect" concept would depend, to a large extent on the virulence of the pathogen as insects, due to the large size of F. tumidum macroconidia, can carry only a small number of it.