Ecological and genetic determinants of Pepino mosaic virus emergence

Virus emergence is a complex phenomenon, which generally involves spread to a new host from a wild host, followed by adaptation to the new host. Although viruses account for the largest fraction of emerging crop pathogens, knowledge about their emergence is incomplete. We address here the question of whether Pepino mosaic virus (PepMV) emergence as a major tomato pathogen worldwide could have involved spread from wild to cultivated plant species and host adaptation. For this, we surveyed natural populations of wild tomatoes in southern Peru for PepMV infection. PepMV incidence, genetic variation, population structure, and accumulation in various hosts were analyzed. PepMV incidence in wild tomatoes was high, and a strain not yet reported in domestic tomato was characterized. This strain had a wide host range within the Solanaceae, multiplying efficiently in most assayed Solanum species and being adapted to wild tomato hosts. Conversely, PepMV isolates from tomato crops showed evidence of adaptation to domestic tomato, possibly traded against adaptation to wild tomatoes. Phylogenetic reconstructions indicated that the most probable ancestral sequence came from a wild Solanum species. A high incidence of PepMV in wild tomato relatives would favor virus spread to crops and its efficient multiplication in different Solanum species, including tomato, allowing its establishment as an epidemic pathogen. Later, adaptation to tomato, traded off against adaptation to other Solanum species, would isolate tomato populations from those in other hosts.

Compromisos en la adaptación a distintos huéspedes en virus de plantas

Los patógenos han desarrollado estrategias para sobrevivir en su entorno, infectar a sus huéspedes, multiplicarse dentro de estos y posteriormente transmitirse a otros huéspedes. Todos estos componentes hacen parte de la eficacia biológica de los patógenos, y les permiten ser los causantes de enfermedades infecciosas tanto en hombres y animales, como en plantas. El proceso de infección produce efectos negativos en la eficacia biológica del huésped y la gravedad de los efectos, dependerá de la virulencia del patógeno. Por su parte, el huésped ha desarrollado mecanismos de respuesta en contra del patógeno, tales como la resistencia, por la que reduce la multiplicación del patógeno, o la tolerancia, por la que disminuye el efecto negativo de la infección. Estas respuestas del huésped a la infección producen efectos negativos en la eficacia biológica del patógeno, actuando como una presión selectiva sobre su población. Si la presión selectiva sobre el patógeno varía según el huésped, se predice que un mismo patógeno no podrá aumentar su eficacia biológica en distintos huéspedes y estará más adaptado a un huésped y menos a otro, disminuyendo su gama de huéspedes. Esto supone que la adaptación de un patógeno a distintos huéspedes estará a menudo dificultada por compromisos (trade-off) en diferentes componentes de la eficacia biológica del patógeno. Hasta el momento, la evidencia de compromisos de la adaptación del patógeno a distintos huéspedes no es muy abundante, en lo que se respecta a los virus de plantas. En las últimas décadas, se ha descrito un aumento en la incidencia de virus nuevos o previamente descritos que producen enfermedades infecciosas con mayor gravedad y/o diferente patogenicidad, como la infección de huéspedes previamente resistentes. Esto se conoce como la emergencia de enfermedades infecciosas y está causada por patógenos emergentes, que proceden de un huésped reservorio donde se encuentran adaptados. Los huéspedes que actúan como reservorios pueden ser plantas silvestres, que a menudo presentan pocos síntomas o muy leves a pesar de estar infectados con diferentes virus, y asimismo se encuentran en ecosistemas con ninguna o poca intervención humana. El estudio de los factores ecológicos y biológicos que actúan en el proceso de la emergencia de enfermedades infecciosas, ayudará a entender sus causas para crear estrategias de prevención y control. Los virus son los principales patógenos causales de la emergencia de enfermedades infecciosas en humanos, animales y plantas y un buen modelo para entender los procesos de la emergencia. Asimismo, las plantas a diferencia de los animales, son huéspedes fáciles de manipular y los virus que las afectan, más seguros para el trabajo en laboratorio que los virus de humanos y animales, otros modelos también usados en la investigación. Por lo tanto, la interacción virus – planta es un buen modelo experimental para el estudio de la emergencia de enfermedades infecciosas. El estudio de la emergencia de virus en plantas tiene también un interés particular, debido a que los virus pueden ocasionar pérdidas económicas en los cultivos agrícolas y poner en riesgo la durabilidad de la resistencia de plantas mejoradas, lo que supone un riesgo en la seguridad alimentaria con impactos importantes en la sociedad, comparables con las enfermedades infecciosas de humanos y animales domésticos. Para que un virus se convierta en un patógeno emergente debe primero saltar desde su huésped reservorio a un nuevo huésped, segundo adaptarse al nuevo huésped hasta que la infección dentro de la población de éste se vuelva independiente del reservorio y finalmente debe cambiar su epidemiología. En este estudio, se escogió la emergencia del virus del mosaico del pepino dulce (PepMV) en el tomate, como modelo experimental para estudiar la emergencia de un virus en una nueva especie de huésped, así como las infecciones de distintos genotipos del virus del moteado atenuado del pimiento (PMMoV) en pimiento, para estudiar la emergencia de un virus que aumenta su patogenicidad en un huésped previamente resistente. El estudio de ambos patosistemas nos permitió ampliar el conocimiento sobre los factores ecológicos y evolutivos en las dos primeras fases de la emergencia de enfermedades virales en plantas. El PepMV es un patógeno emergente en cultivos de tomate (Solanum lycopersicum) a nivel mundial, que se describió primero en 1980 infectando pepino dulce (Solanum muricatum L.) en Perú, y casi una década después causando una epidemia en cultivos de tomate en Holanda. La introducción a Europa posiblemente fue a través de semillas infectadas de tomate procedentes de Perú, y desde entonces se han descrito nuevos aislados que se agrupan en cuatro cepas (EU, LP, CH2, US1) que infectan a tomate. Sin embargo, el proceso de su emergencia desde pepino dulce hasta tomate es un interrogante de gran interés, porque es uno de los virus emergentes más recientes y de gran importancia económica. Para la emergencia de PepMV en tomate, se recolectaron muestras de tomate silvestre procedentes del sur de Perú, se analizó la presencia y diversidad de aislados de PepMV y se caracterizaron tanto biológicamente (gama de huéspedes), como genéticamente (secuencias genomicas). Se han descrito en diferentes regiones del mundo aislados de PMMoV que han adquirido la capacidad de infectar variedades previamente resistentes de pimiento (Capsicum spp), es decir, un típico caso de emergencia de virus que implica la ampliación de su gama de huéspedes y un aumento de patogenicidad. Esto tiene gran interés, ya que compromete el uso de variedades resistentes obtenidas por mejora genética, que es la forma de control de virus más eficaz que existe. Para estudiar la emergencia de genotipos altamente patogénicos de PMMoV, se analizaron clones biológicos de PMMoV procedentes de aislados de campo cuya patogenicidad era conocida (P1,2) y por mutagénesis se les aumentó la patogenicidad (P1,2,3 y P1,2,3,4), introduciendo las mutaciones descritas como responsables de estos fenotipos. Se analizó si el aumento de la patogenicidad conlleva un compromiso en la eficacia biológica de los genotipos de PMMoV. Para ello se evaluaron diferentes componentes de la eficacia biológica del virus en diferentes huéspedes con distintos alelos de resistencia. Los resultados de esta tesis demuestran: i). El potencial de las plantas silvestres como reservorios de virus emergentes, en este caso tomates silvestres del sur de Perú, así como la existencia en estas plantas de aislados de PepMV de una nueva cepa no descrita que llamamos PES. ii) El aumento de la gama de huéspedes no es una condición estricta para la emergencia de los virus de plantas. iii) La adaptación es el mecanismo más probable en la emergencia de PepMV en tomate cultivado. iv) El aumento de la patogenicidad tiene un efecto pleiotrópico en distintos componentes de la eficacia biológica, así mismo el signo y magnitud de este efecto dependerá del genotipo del virus, del huésped y de la interacción de estos factores. ABSTRACT host Pathogens have evolved strategies to survive in their environment, infecting their hosts, multiplying inside them and being transmitted to other hosts. All of these components form part of the pathogen fitness, and allow them to be the cause of infectious diseases in humans, animals, and plants. The infection process produces negative effects on the host fitness and the effects severity will depend on the pathogen virulence. On the other hand, hosts have developed response mechanisms against pathogens such as resistance, which reduces the growth of pathogens, or tolerance, which decreases the negative effects of infection. T he se responses of s to infection cause negative effects on the pathogen fitness, acting as a selective pressure on its population. If the selective pressures on pathogens va ry according to the host s , probably one pathogen cannot increase its fitness in different hosts and will be more adapted to one host and less to another, decreasing its host range. This means that the adaptation of one pathogen to different hosts , will be often limited by different trade - off components of biological effectiveness of pathogen. Nowadays , trade - off evidence of pathogen adaptation to different hosts is not extensive, in relation with plant viruses. In last decades, an increase in the incidence of new or previously detected viruses has been described, causing infectious diseases with increased severity and/or different pathogenicity, such as the hosts infection previously resistants. This is known as the emergence of infectious diseases and is caused by emerging pathogens that come from a reservoir host where they are adapted. The hosts which act as reservoirs can be wild plants, that often have few symptoms or very mild , despite of being infected with different viruses, and being found in ecosystems with little or any human intervention. The study of ecological and biological factors , acting in the process of the infectious diseases emergence will help to understand its causes to create strategies for its prevention and control. Viruses are the main causative pathogens of the infectious diseases emergence in humans, animals and plants, and a good model to understand the emergency processes. Likewise, plants in contrast to animals are easy host to handle and viruses that affect them, safer for laboratory work than viruses of humans and animals, another models used in research. Therefore, the interaction plant-virus is a good experimental model for the study of the infectious diseases emergence. The study of virus emergence in plants also has a particular interest, because the viruses can cause economic losses in agricultural crops and threaten the resistance durability of improved plants, it suppose a risk for food security with significant impacts on society, comparable with infectious diseases of humans and domestic animals. To become an emerging pathogen, a virus must jump first from its reservoir host to a new host, then adapt to a new host until the infection within the population becomes independent from the reservoir, and finally must change its epidemiology. In this study, the emergence of pepino mosaic virus (PepMV) in tomato, was selected as experimental model to study the emergence of a virus in a new host specie, as well as the infections of different genotypes of pepper mild mottle virus (PMMoV) in pepper, to study the emergence of a virus that increases its pathogenicity in a previously resistant host. The study of both Pathosystems increased our knowledge about the ecological and evolutionary factors in the two first phases of the emergence of viral diseases in plants. The PepMV is an emerging pathogen in tomato (Solanum lycopersicum L.) in the world, which was first described in 1980 by infecting pepino (Solanum muricatum L.) in Peru, and almost after a decade caused an epidemic in tomato crops in Netherlands. The introduction to Europe was possibly through infected tomato seeds from Peru, and from then have been described new isolates that are grouped in four strains (EU, LP, CH2, US1) that infect tomato. However, the process of its emergence from pepino up tomato is a very interesting question, because it is one of the newest emerging viruses and economically important. For the PepMV emergence in tomato, wild tomato samples from southern Peru were collected, and the presence and diversity of PepMV isolates were analyzed and characterized at biological (host range) and genetics (genomic sequences) levels. Isolates from PMMoV have been described in different world regions which have acquired the ability to infect pepper varieties that were previously resistants (Capsicum spp), it means, a typical case of virus emergence which involves the host range extension and an increased pathogenicity. This is of great interest due to involve the use of resistant varieties obtained by breeding, which is the most effective way to control virus. To study the emergence of highly pathogenic genotypes of PMMoV, biological clones from field isolates whose pathogenicity was known were analyzed (P1,2) and by mutagenesis we increased its pathogenicity (P1,2,3 and P1,2, 3,4), introducing the mutations described as responsible for these phenotypes. We analyzed whether the increased pathogenicity involves a trade-off in fitness of PMMoV genotypes. For this aim, different components of virus fitness in different hosts with several resistance alleles were evaluated. The results of this thesis show: i). The potential of wild plants as reservoirs of emerging viruses, in this case wild tomatoes in southern Peru, and the existence in these plants of PepMV isolates of a new undescribed strain that we call PES. ii) The host range expansion is not a strict condition for the plant virus emergence. iii) The adaptation is the most likely mechanism in the PepMV emergence in cultivated tomato. iv) The increased pathogenicity has a pleiotropic effect on several fitness components, besides the sign and magnitude of this effect depends on the virus genotype, the host and the interaction of both.

A herpes simplex virus 1 recombinant lacking the glycoprotein G coding sequences is defective in entry through apical surfaces of polarized epithelial cells in culture and in vivo

During infection of a new host, the first surfaces encountered by herpes simplex viruses are the apical membranes of epithelial cells of mucosal surfaces. These cells are highly polarized, and the protein composition of their apical and basolateral membranes are very different, so that different viral entry pathways have evolved for each surface. To determine whether the viral glycoprotein G (gG) is specifically required for efficient infection of a particular surface of polarized cells, apical and basal surfaces were infected with wild-type virus or a gG deletion mutant. After infection of polarized cells in culture, the gG− virus was deficient in infection of apical surfaces but was able to infect cells through basal membranes, replicate, and spread into surrounding cells. The gG-dependent step in apical infection was a stage beyond attachment. After in vivo infection of apical surfaces of epithelial cells of nonscarified mouse corneas, infection by glycoprotein C− or gG− virus was considerably reduced as compared with that observed after infection with wild-type virus. In contrast, when corneas were scarified, allowing virus access to other cell surfaces, the gG and glycoprotein C deletion mutants infected eyes as efficiently as wild-type viruses. A secondary mutation allowing infection of apical surfaces by gG− virus arose readily during passage of the virus in nonpolarized cells, indicating that either the gG-dependent step of apical infection can be bypassed or that another viral protein can acquire the same function.

Positive selection and rates of evolution in immunodeficiency viruses from humans and chimpanzees.

Evolutionary theory predicts the recent spread of primate immunodeficiency viruses (PIVs) to new human populations to be accompanied by positive selection in response to new host environments and/or by random genetic drift. I assess evidence for positive selection in human and chimpanzee PIVs type I (PIV1s), using ratios of synonymous to nonsynonymous nucleotide change based on branch lengths and outgroup rooting. Ratios are smaller for PIV1s from humans than for PIV1 from a chimpanzee for the pol, gag, and env glycoprotein 120 (gp120) regions, indicating greater effects of positive selection in PIV1s from humans. Parsimony-based relative rate tests for amino acid changes showed significant differences between PIV1s from humans and chimpanzees in 18 of 48 pairwise comparisons, with all 18 showing faster rates of change in PIV1s from humans. This study indicates that in some instances, the recent evolution of human PIV1s follows a speciational pattern, in which increased diversification of taxa is correlated with greater amounts of character change appearing and being maintained through time. This extends the generality of the speciational pattern to a group of organisms (viruses) having the fastest known rates of anagenetic change for nucleotide characters and indicates that comprehensive understanding of PIV1 evolution requires consideration of both anagenetic change within viral lineages and the relative historical success of different viral clades. Phylogenetic analyses show that neither PIV1s infecting humans nor those infecting chimpanzees represent monophyletic groups and suggest multiple host-species shifts for PIV1s.

Multiple origins of the yucca-yucca moth association.

The association of species of yucca and their pollinating moths is considered one of the two classic cases of obligate mutualism between floral hosts and their pollinators. The system involves the active collection of pollen by females of two prodoxid moth genera and the subsequent purposeful placement of the pollen on conspecific stigmas of species of Yucca. Yuccas essentially depend on the moths for pollination and the moths require Yucca ovaries for oviposition. Because of the specificity involved, it has been assumed that the association arose once, although it has been suggested that within the prodoxid moths as a whole, pollinators have arisen from seed predators more than once. We show, by using phylogenies generated from three molecular data sets, that the supposed restriction of the yucca moths and their allies to the Agavaceae is an artifact caused by an incorrect circumscription of this family. In addition we provide evidence that Yucca is not monophyletic, leading to the conclusion that the modern Yucca-yucca moth relationship developed independently more than once by colonization of a new host.

The ectoparasitic mite Tropilaelaps mercedesae reduces western honey bee, Apis mellifera, longevity and emergence weight, and promotes Deformed wing virus infections

Historically an ectoparasite of the native Giant honey bee Apis dorsata, the mite Tropilaelaps mercedesae has switched hosts to the introduced western honey bee Apis mellifera throughout much of Asia. Few data regarding lethal and sub-lethal effects of T. mercedesae on A. mellifera exist, despite its similarity to the devastating mite Varroa destructor. Here we artificially infested worker brood of A. mellifera with T. mercedesae to investigate lethal (longevity) and sub-lethal (emergence weight, Deformed wing virus (DWV) levels and clinical symptoms of DWV) effects of the mite on its new host. The data show that T. mercedesae infestation significantly reduced host longevity and emergence weight, and promoted both DWV levels and associated clinical symptoms. Our results suggest that T. mercedesae is a potentially important parasite to the economically important A. mellifera honey bee.

Multifaceted roles of the lysosome in the establishment and expansion of tuberculous infection

Thesis (Ph.D.)--University of Washington, 2016-04

Patterns of digenean parasitism of bivalves from the Great Barrier Reef and associated waters

Digenean parasites of marine bivalves are relatively poorly known, particularly in Australia. We surveyed 2256 bivalve individuals ( 47 species, 17 families) from Queensland marine waters incorporating south-east Queensland, Heron Island ( southern Great Barrier Reef) and Lizard Island ( northern Great Barrier Reef). Infections of trematode species from three families, Bucephalidae, Gorgoderidae and Monorchiidae, were found. Overall prevalence of infection was 2.3%. The Bucephalidae was the most commonly found family; 11 species were found in Tellinidae, Ostreidae, Isognomonidae and Spondylidae - the latter two previously unknown as hosts for bucephalids. A single gorgoderid infection was found in a venerid, Lioconcha castrensis. Five species of monorchiids were found from Tellinidae and Lucinidae. All infections are new host/parasite records. No infections were found in 35 of the 47 bivalve species sampled. The generally low prevalence of infection by digeneans of bivalves suggests that it is unlikely that any of the species reported here are seriously damaging to bivalve populations in these waters. We deduce that, at best, we have some life-cycle information but no actual identifications for 10% of the species of trematodes that infect bivalves of Queensland marine waters.

Trichodinids (Ciliophora : Trichodinidae) from native and exotic Australian freshwater fishes

There are very few data on trichodinids of freshwater fishes in Australia. 2003 fishes were surveyed across Eastern Australia to investigate the diversity of trichodinids present, to determine which species have been introduced with exotic fishes and to determine the extent to which these species have crossed into native fish Populations. Twenty-one putative trichodinid species were recovered from the 33 fish species examined. Trichodina heterodentata, T. mutabilis and T. reticulata were the exotic species recovered regularly; a single specimen matched a fourth exotic species, T acuta. All four exotic species are redescribed from Australian material. Trichodina heterodentata was recorded from 17 species of fishes, 15 of which were new host records; this species is identified as one of emerging importance in fish parasitology and a list of its known hosts is presented. Two new native species are also described based on silver stained specimens: T cribbi sp. n. from Hypseleotris galii, H. klunzingeri, and Hypseleotris sp. 5; and T. bassonae sp. n. from Selenotoca multifasciata. Trichodina cribbi is characterised by a large circular central inclusion and approximately 28 denticles, which have a blade length slightly greater than the ray length. Trichodina bassonae is characterised by a small, round, central inclusion and approximately 25 denticles, which have straight, non tapering rays that are in line with the leading edge of the denticle blade. It is estimated that the Australian trichodinid fauna may include up to 150 as yet undescribed species and represents a major source of unexplored biodiversity.

Thelastomatoidea (Nematoda : Oxyurida) of the Australian giant burrowing cockroach, Macropanesthia rhinoceros (Blaberidae : Geoscapheinae)

The thelastomatoid fauna of Macropanesthia rhinoceros was examined from 13 localities across its range in Queensland, Australia. Nine species of thelastomatoids, including two representing new genera, Geoscaphenema megaovum n. g., n. sp. and Jaidenema rhinoceratum n. g., n. sp., were found. Macropanesthia rhinoceros is reported as a new host for seven species previously recorded from Panesthia cribrata (Blaberidae: Panesthiinae) and P. tryoni tryoni, viz, Blattophila sphaerolaima, Leidynemella fusiformis, Cordonicola gibsoni, Travassosinema jaidenae, Coronostoma australiae, Hammerschmidtiella hochi and Desmicola ornata. Overall estimated richness for the system ranged from 10.1-13.5 species. The high degree of parasite faunal overlap between M. rhinoceros and the two Panesthia species is surprising given the disparate ecological niches that they occupy; P. cribrata and P. tryoni tryoni burrow in, and feed upon, moist decaying wood and require a climate that is moist all year round, whereas M. rhinoceros burrows in loose soil, feeds on fallen leaf litter and is tolerant of much drier environments.

Hepatocyte determinants of susceptibility to pre-erythrocytic Plasmodium infection

Thesis (Ph.D.)--University of Washington, 2016-08

Host selection in phytophagous insects: a new explanation for learning in adults

Insect learning can change the preferences an egg laying female displays towards different host plant species. Current hypotheses propose that learning may be advantageous in adult host selection behaviour through improved recognition, accuracy or selectivity in foraging. In this paper, we present a hypothesis for when learning can be advantageous without such improvements in adult host foraging. Specifically, that learning can be an advantageous strategy for egg laying females when larvae must feed on more than one plant in order to complete development, if the fitness of larvae is reduced when they switch to a different host species. Here, larvae benefit from developing on the most abundant host species, which is the most likely choice of host for an adult insect which increases its preference for a host species through learning. The hypothesis is formalised with a mathematical model and we provide evidence from studies on the behavioural ecology, of a number of insect species which demonstrate that the assumptions of this hypothesis may frequently be fulfilled in nature. We discuss how multiple mechanisms may convey advantages in insect learning and that benefits to larval development, which have so far been overlooked, should be considered in explanations for the widespread occurrence of learning.

Mosquito host-feeding patterns and implications for Japanese encephalitis virus transmission in northern Australia and Papua New Guinea

Japanese encephalitis (JE) virus spread to northern Australia during the 1990s, transmitted by Culex annulirostris Skuse and other mosquitoes (Diptera: Culicidae). To determine the relative importance of various hosts for potential vectors of JE virus, we investigated the host-feeding patterns of mosquitoes in northern Australia and Western Province of Papua New Guinea, with particular attention to pigs, Sus scrofa L. - the main amplifying host of JE virus in South-east Asia. Mosquitoes were collected by CDC light traps baited with dry ice and 1-octen-3-ol, run 16.00-08.00 hours, mostly set away from human habitations, if possible in places frequented by feral pigs. Bloodmeals of 2569 mosquitoes, representing 15 species, were identified by gel diffusion assay. All species had fed mostly on mammals: only 30%) were trapped where domestic pigs were kept close to human habitation. From seven of eight locations on the Australian mainland, the majority of Cx. annulirostris had obtained their bloodmeals from marsupials, probably the Agile wallaby Macropus agilis (Gould). Overall proportions of mosquito bloodmeals identified as marsupial were 60% from the Gulf Plains region of Australia, 78% from the Cape York Peninsula and 64% from the Daru area of Papua New Guinea. Thus, despite the abundance of feral pigs in northern Australia, our findings suggest that marsupials divert host-seeking Cx. annulirostris away from pigs. As marsupials are poor JE virus hosts, the prevalence of marsupials may impede the establishment of JE virus in Australia.

New insights into host-parasite ubiquitin proteome dynamics in P. falciparum infected red blood cells using a TUBEs-MS approach

Malaria, caused by Plasmodium falciparum (P. falciparum), ranks as one of the most baleful infectious diseases worldwide. New antimalarial treatments are needed to face existing or emerging drug resistant strains. Protein degradation appears to play a significant role during the asexual intraerythrocytic developmental cycle (IDC) of P. falciparum. Inhibition of the ubiquitin proteasome system (UPS), a major intracellular proteolytic pathway, effectively reduces infection and parasite replication. P. falciparum and erythrocyte UPS coexist during IDC but the nature of their relationship is largely unknown. We used an approach based on Tandem Ubiquitin-Binding Entities (TUBEs) and 1D gel electrophoresis followed by mass spectrometry to identify major components of the TUBEs-associated ubiquitin proteome of both host and parasite during ring, trophozoite and schizont stages. Ring-exported protein (REX1), a P. falciparum protein located in Maurer's clefts and important for parasite nutrient import, was found to reach a maximum level of ubiquitylation in trophozoites stage. The Homo sapiens (H. sapiens) TUBEs associated ubiquitin proteome decreased during the infection, whereas the equivalent P. falciparum TUBEs-associated ubiquitin proteome counterpart increased. Major cellular processes such as DNA repair, replication, stress response, vesicular transport and catabolic events appear to be regulated by ubiquitylation along the IDC P. falciparum infection.

Mediterranean spotted fever and identification of new agents of rickettsioses in Portugal : epidemiological determinants, host and microbial features in portuguese patients

Resumo: a febre botonosa, também conhecida por febre escaro-nodular (FEN) é uma doença endémica nos Países da bacia do Mediterrâneo, África, Médio Oriente, Índia e Paquistão. O agente etiológico responsável por esta patologia é a bactéria Rickettsia conorii. Contudo, em alguns países, como Portugal e Itália, esta patologia é causada por duas estirpes diferentes: R conorii Malish e R conorii Israeli spotted fever strain. O principal vector e reservatório é o ixodídeo Rhipicephalus sanguineus. Mesmo com uma elevada taxa de subnotificação detectada no nosso País, a taxa incidência da FEN é de 8.4/105 habitantes (1989-2005), uma das mais altas quando comparada coom a de outros países da bacia do Mediterrâneo. De todos os distritos portugueses, Bragança e Beja são aqueles que apresentam as taxas de incidência mais elevadas, 56,8/105 habitantes e 47,4 / 105 habitantes respectivamente. Em Portugal, as alterações climáticas verificadas na última década, nomeadamente a subida das temperaturas médias anuais, parecem ter influenciado o ciclo de vida do vector e a sua dinâmica sazonal, permitindo ao R. sanguineus completar mais de um ciclo de vida por ano. Este facto, e a possibilidade deste vector se manter activo noutros meses do ano, nomeadamente nos meses de inverno, tem influenciado consequentemente o padrão de distribuição anual dos casos de FEN. A febre escaro-nodular caracteriza-se clinicamente como uma doença exantemática, com um processo de vasculite generalizado. Apesar de na generalidade ser considerada uma doença benigna (quando tratada atempadamente e com terapêutica adequada e específica)e de estarem descritos casos graves em cerca de 5-6% dos doentes, em Portugal essa percentagem aumentou e consequentemente levou a um aumento de casos fatais. Este facto tornou-se mais evidente em 1997, no Hospital Distrital de Beja e no Hospital Garcia de Orta, onde a taxa de letalidade atingiu os 32% e 18% respectivamente.Para além dos factores de co-morbilidade encontrados nos doentes mais graves, como diabetes mellitus, ou o atraso na instituição da terapêutica específica, foi colocada de que a estirpe R. conorii Israel spotted fever strain pudesse ser mais virulenta ou então estivesse associada a diferentes manifestações clínicas que dificultassem o diagnóstico clínico e a instituição atempada da terapêutica. Houve ainda a necessidade de avaliar alguns parâmetros imunológicos dos doentes e tentar identificar que factores, nomeadamente que citoquinas, poderiam estar envolvidos na resposta a uma infecção por R.conorii.Face a estas questões foi avaliada e comparada a epidemiologia, manifestações clínicas e laboratoriais de 140 doentes (71 infectados com R. conorii Malish e 69 infectados com R. conorii Israel spotted fever strain). Concluiu-se que existe uma sobreposição de manifestações clinicas entre os dois grupos de doentes, mas que a percentagem da escara de inoculação é significativamente inferior em doentes infectados com R. conorii Israel spotted fever strain. Dos resultados mais importantes encontrados neste estudo concluiu-se que a estirpe R. conorii Malish e é demonstrado, pela primeira vez, estatisticamente que o alcoolismo é um factor de risco para a morte de doentes com FEN. Associadas a factores de um mau prognósitco da doença, estão as manifestações gastrointestinais, que poderão ser ou não reflexo de alterações do sistema nervoso central, e ainda a alteração de parâmetros laboratoriais como a presença de hiperbilirubinemia e aumento dos valores da ureia.A maior parte dos estudos realizados sobre os mecanismos da resposta imunitária à infecção por R. conorii e as interacções hospedeiro - agente etiológico têm sido elucidados com base em modelos animais. Poucos estudos têm sido efectuados em doentes e nenhum estudo prévio tinha sido realizado no sentido de avaliar localmente (escara/pele) quais os mediadores ou outras moléculas envolvidas na resposta imunitária às rickettsioses. Foi avaliado o nível de expressão génica de RNA mensageiro (RNAm)de diferentes citoquinas em amostras de pele de doentes com FEN pela técnica de PCR em tempo real.Os resultados deste estudo mostraram que, quando comparado com o grupo controlo, os 23 doentes analisados apresentavam níveis estatisticamente significativos, mais elevados de expressão génica de interferão (IFN-γ, Tumor necrosis factor (TFN-α, interleucina 10 (IL-10, RANTES (regulated by activation, normal T-cell-expressed and secreted chemokine)e indolamina 2-3 desoxigenase (IDO),uma enzima envolvida no controlo e limitação do crescimento intracelular das rickettsias, através da degradação do triptofano. Seis dos 23 doentes apresentaram ainda niveis de expressão elevados de óxido nítrico indutível (iNOS)que actua como microbicida. Encontrou-se uma correlação positiva entre a expressão de RNAm de TNF-α, γ, iNOS e IDO e os casos menos graves de FEN sugerindo um tipo de resposta imunitária tipo Th1, i.e. com papel protector na resposta à infecção.Verificou-se também que os valores de expressão genética do RNAm de IL-10, estavam inversamente correlacionados com a expressão do RNAm de TNF-α e IFN-γ. Os casos menos graves de FEN parecem assim envolver um balanço entre a resposta pró-inflamatória e anti-inflamatória. Já os níveis de expressão génica do RNAm de IL-10 estavam inversamente correlacionados com a expressão RNAm de TNF-α e IFN-γ. Os casos menos graves de FEN parecem assim envolver um balanço entre uma resposta pró-inflamatória e anti-inflamatória. Já os níveis de expressão RNAm da quimoquina RANTES foram estatisticamente mais elevados em doentes graves.Nesta dissertação é ainda descrita uma nova rickettsiose presente em Portugal, causada pela bactéria R. sibirica mongolitimonae, que foi identificada laboratorialmente por isolamento do agente, e por detecção do DNA em biopsia de pele. A presença deste agente foi ainda corroborada pela detecção em paralelo do mesmo agente no ixodídeos como R. africae like e em pulgas como R. felis e R.typhi alertam para a possibilidade de existência de outras rickettsioses que possam estar diagnosticadas em Portugal. Abstract: Mediterranean spotted fever (MSF), a tick-borne disease caused by Rickettsia conorii, is widley distributed in the Old World, being endemic in the southern Europe, Africa, Middle East, India and Pakistan. In Portugal two strains cause disease: R.conorii Malish and R.conorii Israeli spotted fever.Rhipicephalus sanguineus, the brown dog tick, is considered the main vector and reservoir. MSF is characterized by seasonality, and most of cases are encountered in late spring and summer, peaking in July and August. However, CEVDI/INSA laboratory has observed that the incidence of MSF cases has changed during winter season.The increasing annual averages of air temperatures and warmer and drier winters might have influenced the dynamics of the life cycle and activity of R. sanguineus, and indirectley the number MSF cases during the so called MSF off-season.In the period of 1989-2005, the incidence rate of MSF was 8.4/105 inhabitants, one of the highest rates compared with other endemic countries. In the Portugal during the same period, the highest incidence rates were reported in the districts of Bragança, with 56.8/105 inhabitants, and Beja, with 47.4/105 inhabitants. Severe cases of MSF are reported in 6% of the patients, but it seems that this pattern of disease in Portugal has been changing.This factor became more evident in 1997, with a reported case fatality rate of 32% and 18% in patients with MSF admited at Beja and Garcia Orta Hospitals, respectively. Although it was found that diabetes mellitus and delay in therapy have been implicated as a risk factor for death, the hypothesis was considered, that the new ISF strain isolated from Portugueses patients in the same year (1997)causes different or atypical clinical conorii Malish strain. The local (skin biopsies) immune response to R. conorii infection was also evaluated.A prospective study was performed to characterized epidemiological, clinical, laboratory features and determined risk factors for a fatal outcome. One hundred forty patients (51% patients were infected with Rickettsia conorii Malish stain and 49% with Israeli spotted fever strain)with diagnosis documented with identification of the causative rickettsial strain were admitted to 13 Portugueses Hospitals during 1994-2006.Comparison of the clinical manifestations of MSF caused by Malish and ISF strains revealed tremendous overlap that would not permit clinical recognition of the strain envolved, but an eschar was observed in a significantly higher percentage of patients with Malish than ISF strain.A fatal outcome was significantly more likely for patients with ISF strain infection meaning that ISF strain was more virulent than Malish strain, and also alcoholism was a host risk factor for a fatal outcome.The pathophysiology of a fatal outcome involved significantly greater incidence of petechial rash, gastrointestinal symptoms, confusion/obtundation, dehydration, tachypnea, hepatomegaly, leukocytosis, coagulopathy, azotemia, hyperbilirubinemia, and elevated hepatic enzymes and creatine kinase. Multivariate analysis revealed that acute renal failure and hyperbilirubinemia were most strong associated with a fatal oucome of infections with both strains.The immune response to R. conorii infection determined with both strains. The immune response to R. conorii infection determined by the expression levels of inflammatory and immune mediators in skin biopsies collected from untreated patients with Mediterranean spotted fever reveal that intralesional expression of mRNA of TNF-α, IFN-γ, IL-10, RANTES, and indoleamine-2, 3-dioxygenase (IDO)an enzyme involved in limiting rickettsial growth by tryptophan degradation, were elevated in skin of MSF patients compared to controls. Six patients had elevated levels of inducible nitric oxide synthase (NOS2, a source microbicidal nitric oxide.Positive correlations among TNF-α, IFN-γ, NOS2,IDO and mild-to-moderate disease suggested that type 1 polarization plays a protective role. Significantly high levels of intralesional IL-10 were inversely correlated with IFN-γ and TNF-α. The chemokine RANTES was significantly higher in patients with several MSF. It seems that MSF patients with mild-to-moderate disease have a strong and balanced intralesional pro-inflammatory and anti-inflammatory response, while severe disease is associated with higher chemokine expression.Whether these findings are simply a correlate of mild and severe disease or contribute to anti-rickettsial immunity and pathogenesis remains to be determined.In this dissertation is also described a new rickettsiois present in Portugal caused by R.sibirica mongolitimonae strain, identified based on agent isolation and DNA detection by PCR technique in a skin biopsy.The presence of this agent corroborated by its detection also in Rhipicephalus pusillus tick. Also, pathogenic tick and flea-borne rickettsial agents such as R. africae strain detected in Rhipicephalus bursa tick, and R.felis and R.typhi detected in different fleas species raise the alert for the possible existence of other rickettsioses in Portugal that might be underdiagnosed.