Expressão em Escherichia coli e produção de antissoro policlonal para proteína P1 do Southern bean mosaic virus

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

The receptor-like kinase SlSOBIR1 is differentially modulated by virus infection but its overexpression in tobacco has no significant impact on virus accumulation

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Avaliação da produção e concentração viral em pimentão infectado por tospovírus com o uso de piraclostrobina + metiram

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Epidemiological and molecular characterization of rubella virus isolated in Sao Paulo, Brazil during 1997-2004

Rubella virus (RV) infection during the early stages of pregnancy can lead to serious birth defects, known as the congenital rubella syndrome (CRS). In 2003, the Pan American Health Organization (PAHO) adopted a resolution calling for the elimination of rubella and the congenital rubella syndrome (CRS) in the Americas by the year 2010. Brazil will have implemented the recommended PAHO strategy for elimination and interruption of endemic rubella virus transmission. The characterization of genotypes during the final stages of rubella elimination is important for determining whether new rubella isolates represent endemic transmission or importations. Samples (blood, urine, cerebrospinal fluid, and throat swabs) collected from patients with symptoms suggestive of rubella infection in 19972004 were isolated in cell culture and genotyped. Twenty-eight sequences were analyzed and two genotypes were identified: 1a and 1G. The information reported in this paper will contribute to understanding the molecular epidemiology of RV in Sao Paulo, Brazil. J. Med. Virol. 84:18311838, 2012. (c) 2012 Wiley Periodicals, Inc.

Dengue transmission risk maps of the continental United States

Dengue fever is a strictly human and non-human primate disease characterized by a high fever, thrombocytopenia, retro-orbital pain, and severe joint and muscle pain. Over 40% of the world population is at risk. Recent re-emergence of dengue outbreaks in Texas and Florida following the re-introduction of competent Aedes mosquito vectors in the United States have raised growing concerns about the potential for increased occurrences of dengue fever outbreaks throughout the southern United States. Current deficiencies in vector control, active surveillance and awareness among medical practitioners may contribute to a delay in recognizing and controlling a dengue virus outbreak. Previous studies have shown links between low-income census tracts, high population density, and dengue fever within the United States. Areas of low-income and high population density that correlate with the distribution of Aedes mosquitoes result in higher potential for outbreaks. In this retrospective ecologic study, nine maps were generated to model U.S. census tracts’ potential to sustain dengue virus transmission if the virus was introduced into the area. Variables in the model included presence of a competent vector in the county and census tract percent poverty and population density. Thirty states, 1,188 counties, and 34,705 census tracts were included in the analysis. Among counties with Aedes mosquito infestation, the census tracts were ranked high, medium, and low risk potential for sustained transmission of the virus. High risk census tracts were identified as areas having the vector, ≥20% poverty, and ≥500 persons per square mile. Census tracts with either ≥20% poverty or ≥500 persons per square mile and have the vector present are considered moderate risk. Census tracts that have the vector present but have <20% poverty and <500 persons per square mile are considered low risk. Furthermore, counties were characterized as moderate risk if 50% or more of the census tracts in that county were rated high or moderate risk, and high risk if 25% or greater were rated high risk. Extreme risk counties, which were primarily concentrated in Texas and Mississippi, were considered having 50% or greater of the census tracts ranked as high risk. Mapping of geographic areas with potential to sustain dengue virus transmission will support surveillance efforts and assist medical personnel in recognizing potential cases. ^

Practice advisory: interim guidance for care of obstetric patients during a zika virus outbreak

Zika during pregnancy has been associated with birth defects, specifically significant microcephaly. Transmission of Zika to the fetus has been documented in all trimesters; Zika virus RNA has been detected in fetal tissue from early missed abortions, amniotic fluid, term neonates and the placenta. However, much is not yet known about Zika virus in pregnancy. Uncertainties include the incidence of Zika virus infection among pregnant women in areas of Zika virus transmission, the rate of vertical transmission and the rate with which infected fetuses manifest complications such as microcephaly or demise. The absence of this important information makes management and decision making in the setting of potential Zika virus exposure (i.e. travel to endemic areas) or maternal infection, difficult. Currently, there is no vaccine or treatment for this infection.

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.

Posttranscriptional Gene Silencing in Transgenic Sugarcane. Dissection of Homology-Dependent Virus Resistance in a Monocot That Has a Complex Polyploid Genome1

RNA-mediated, posttranscriptional gene silencing has been determined as the molecular mechanism underlying transgenic virus resistance in many plant virus-dicot host plant systems. In this paper we show that transgenic virus resistance in sugarcane (Saccharum spp. hybrid) is based on posttranscriptional gene silencing. The resistance is derived from an untranslatable form of the sorghum mosaic potyvirus strain SCH coat protein (CP) gene. Transgenic sugarcane plants challenged with sorghum mosaic potyvirus strain SCH had phenotypes that ranged from fully susceptible to completely resistant, and a recovery phenotype was also observed. Clones derived from the same transformation event or obtained after vegetative propagation could display different levels of virus resistance, suggesting the involvement of a quantitative component in the resistance response. Most resistant plants displayed low or undetectable steady-state CP transgene mRNA levels, although nuclear transcription rates were high. Increased DNA methylation was observed in the transcribed region of the CP transgenes in most of these plants. Collectively, these characteristics indicate that an RNA-mediated, homology-dependent mechanism is at the base of the virus resistance. This work extends posttranscriptional gene silencing and homology-dependent virus resistance, so far observed only in dicots, to an agronomically important, polyploid monocot.

Accurate reconstruction of a known HIV-1 transmission history by phylogenetic tree analysis.

Phylogenetic analyses are increasingly used in attempts to clarify transmission patterns of human immunodeficiency virus type 1 (HIV-1), but there is a continuing discussion about their validity because convergent evolution and transmission of minor HIV variants may obscure epidemiological patterns. Here we have studied a unique HIV-1 transmission cluster consisting of nine infected individuals, for whom the time and direction of each virus transmission was exactly known. Most of the transmissions occurred between 1981 and 1983, and a total of 13 blood samples were obtained approximately 2-12 years later. The p17 gag and env V3 regions of the HIV-1 genome were directly sequenced from uncultured lymphocytes. A true phylogenetic tree was constructed based on the knowledge about when the transmissions had occurred and when the samples were obtained. This complex, known HIV-1 transmission history was compared with reconstructed molecular trees, which were calculated from the DNA sequences by several commonly used phylogenetic inference methods [Fitch-Margoliash, neighbor-joining, minimum-evolution, maximum-likelihood, maximum-parsimony, unweighted pair group method using arithmetic averages (UPGMA), and a Fitch-Margoliash method assuming a molecular clock (KITSCH)]. A majority of the reconstructed trees were good estimates of the true phylogeny; 12 of 13 taxa were correctly positioned in the most accurate trees. The choice of gene fragment was found to be more important than the choice of phylogenetic method and substitution model. However, methods that are sensitive to unequal rates of change performed more poorly (such as UPGMA and KITSCH, which assume a constant molecular clock). The rapidly evolving V3 fragment gave better reconstructions than p17, but a combined data set of both p17 and V3 performed best. The accuracy of the phylogenetic methods justifies their use in HIV-1 research and argues against convergent evolution and selective transmission of certain virus variants.

Association of HIV-1 Envelope-Specific Breast Milk IgA Responses with Reduced Risk of Postnatal Mother-to-Child Transmission of HIV-1.

UNLABELLED: Infants born to HIV-1-infected mothers in resource-limited areas where replacement feeding is unsafe and impractical are repeatedly exposed to HIV-1 throughout breastfeeding. Despite this, the majority of infants do not contract HIV-1 postnatally, even in the absence of maternal antiretroviral therapy. This suggests that immune factors in breast milk of HIV-1-infected mothers help to limit vertical transmission. We compared the HIV-1 envelope-specific breast milk and plasma antibody responses of clade C HIV-1-infected postnatally transmitting and nontransmitting mothers in the control arm of the Malawi-based Breastfeeding Antiretrovirals and Nutrition Study using multivariable logistic regression modeling. We found no association between milk or plasma neutralization activity, antibody-dependent cell-mediated cytotoxicity, or HIV-1 envelope-specific IgG responses and postnatal transmission risk. While the envelope-specific breast milk and plasma IgA responses also did not reach significance in predicting postnatal transmission risk in the primary model after correction for multiple comparisons, subsequent exploratory analysis using two distinct assay methodologies demonstrated that the magnitudes of breast milk total and secretory IgA responses against a consensus HIV-1 envelope gp140 (B.con env03) were associated with reduced postnatal transmission risk. These results suggest a protective role for mucosal HIV-1 envelope-specific IgA responses in the context of postnatal virus transmission. This finding supports further investigations into the mechanisms by which mucosal IgA reduces risk of HIV-1 transmission via breast milk and into immune interventions aimed at enhancing this response. IMPORTANCE: Infants born to HIV-1-infected mothers are repeatedly exposed to the virus in breast milk. Remarkably, the transmission rate is low, suggesting that immune factors in the breast milk of HIV-1-infected mothers help to limit transmission. We compared the antibody responses in plasma and breast milk of HIV-1-transmitting and -nontransmitting mothers to identify responses that correlated with reduced risk of postnatal HIV-1 transmission. We found that neither plasma nor breast milk IgG antibody responses were associated with risk of HIV-1 transmission. In contrast, the magnitudes of the breast milk IgA and secretory IgA responses against HIV-1 envelope proteins were associated with reduced risk of postnatal HIV-1 transmission. The results of this study support further investigations of the mechanisms by which mucosal IgA may reduce the risk of HIV-1 transmission via breastfeeding and the development of strategies to enhance milk envelope-specific IgA responses to reduce mother-to-child HIV transmission and promote an HIV-free generation.

On the optimal capacity expansion of electric power systems

The analysis of investment in the electric power has been the subject of intensive research for many years. The efficient generation and distribution of electrical energy is a difficult task involving the operation of a complex network of facilities, often located over very large geographical regions. Electric power utilities have made use of an enormous range of mathematical models. Some models address time spans which last for a fraction of a second, such as those that deal with lightning strikes on transmission lines while at the other end of the scale there are models which address time horizons consisting of ten or twenty years; these usually involve long range planning issues. This thesis addresses the optimal long term capacity expansion of an interconnected power system. The aim of this study has been to derive a new, long term planning model which recognises the regional differences which exist for energy demand and which are present in the construction and operation of power plant and transmission line equipment. Perhaps the most innovative feature of the new model is the direct inclusion of regional energy demand curves in the nonlinear form. This results in a nonlinear capacity expansion model. After review of the relevant literature, the thesis first develops a model for the optimal operation of a power grid. This model directly incorporates regional demand curves. The model is a nonlinear programming problem containing both integer and continuous variables. A solution algorithm is developed which is based upon a resource decomposition scheme that separates the integer variables from the continuous ones. The decompostion of the operating problem leads to an interactive scheme which employs a mixed integer programming problem, known as the master, to generate trial operating configurations. The optimum operating conditions of each trial configuration is found using a smooth nonlinear programming model. The dual vector recovered from this model is subsequently used by the master to generate the next trial configuration. The solution algorithm progresses until lower and upper bounds converge. A range of numerical experiments are conducted and these experiments are included in the discussion. Using the operating model as a basis, a regional capacity expansion model is then developed. It determines the type, location and capacity of additional power plants and transmission lines, which are required to meet predicted electicity demands. A generalised resource decompostion scheme, similar to that used to solve the operating problem, is employed. The solution algorithm is used to solve a range of test problems and the results of these numerical experiments are reported. Finally, the expansion problem is applied to the Queensland electricity grid in Australia.

On the optimal capacity expansion of electric power systems

The analysis of investment in the electric power has been the subject of intensive research for many years. The efficient generation and distribution of electrical energy is a difficult task involving the operation of a complex network of facilities, often located over very large geographical regions. Electric power utilities have made use of an enormous range of mathematical models. Some models address time spans which last for a fraction of a second, such as those that deal with lightning strikes on transmission lines while at the other end of the scale there are models which address time horizons consisting of ten or twenty years; these usually involve long range planning issues. This thesis addresses the optimal long term capacity expansion of an interconnected power system. The aim of this study has been to derive a new, long term planning model which recognises the regional differences which exist for energy demand and which are present in the construction and operation of power plant and transmission line equipment. Perhaps the most innovative feature of the new model is the direct inclusion of regional energy demand curves in the nonlinear form. This results in a nonlinear capacity expansion model. After review of the relevant literature, the thesis first develops a model for the optimal operation of a power grid. This model directly incorporates regional demand curves. The model is a nonlinear programming problem containing both integer and continuous variables. A solution algorithm is developed which is based upon a resource decomposition scheme that separates the integer variables from the continuous ones. The decompostion of the operating problem leads to an interactive scheme which employs a mixed integer programming problem, known as the master, to generate trial operating configurations. The optimum operating conditions of each trial configuration is found using a smooth nonlinear programming model. The dual vector recovered from this model is subsequently used by the master to generate the next trial configuration. The solution algorithm progresses until lower and upper bounds converge. A range of numerical experiments are conducted and these experiments are included in the discussion. Using the operating model as a basis, a regional capacity expansion model is then developed. It determines the type, location and capacity of additional power plants and transmission lines, which are required to meet predicted electicity demands. A generalised resource decompostion scheme, similar to that used to solve the operating problem, is employed. The solution algorithm is used to solve a range of test problems and the results of these numerical experiments are reported. Finally, the expansion problem is applied to the Queensland electricity grid in Australia

The Prehistory of Potyviruses: Their Initial Radiation Was during the Dawn of Agriculture

Background: Potyviruses are found world wide, are spread by probing aphids and cause considerable crop damage. Potyvirus is one of the two largest plant virus genera and contains about 15% of all named plant virus species. When and why did the potyviruses become so numerous? Here we answer the first question and discuss the other. Methods and Findings: We have inferred the phylogenies of the partial coat protein gene sequences of about 50 potyviruses, and studied in detail the phylogenies of some using various methods and evolutionary models. Their phylogenies have been calibrated using historical isolation and outbreak events: the plum pox virus epidemic which swept through Europe in the 20th century, incursions of potyviruses into Australia after agriculture was established by European colonists, the likely transport of cowpea aphid-borne mosaic virus in cowpea seed from Africa to the Americas with the 16th century slave trade and the similar transport of papaya ringspot virus from India to the Americas. Conclusions/Significance: Our studies indicate that the partial coat protein genes of potyviruses have an evolutionary rate of about 1.1561024 nucleotide substitutions/site/year, and the initial radiation of the potyviruses occurred only about 6,600 years ago, and hence coincided with the dawn of agriculture. We discuss the ways in which agriculture may have triggered the prehistoric emergence of potyviruses and fostered their speciation.

The evolutionary value of recombination is constrained by genome modularity

Genetic recombination is a fundamental evolutionary mechanism promoting biological adaptation. Using engineered recombinants of the small single-stranded DNA plant virus, Maize streak virus (MSV), we experimentally demonstrate that fragments of genetic material only function optimally if they reside within genomes similar to those in which they evolved. The degree of similarity necessary for optimal functionality is correlated with the complexity of intragenomic interaction networks within which genome fragments must function. There is a striking correlation between our experimental results and the types of MSV recombinants that are detectable in nature, indicating that obligatory maintenance of intragenome interaction networks strongly constrains the evolutionary value of recombination for this virus and probably for genomes in general.

Advanced Engineering of Lipid Metabolism in Nicotiana benthamiana Using a Draft Genome and the V2 Viral Silencing-Suppressor Protein

The transient leaf assay in Nicotiana benthamiana is widely used in plant sciences, with one application being the rapid assembly of complex multigene pathways that produce new fatty acid profiles. This rapid and facile assay would be further improved if it were possible to simultaneously overexpress transgenes while accurately silencing endogenes. Here, we report a draft genome resource for N. benthamiana spanning over 75% of the 3.1 Gb haploid genome. This resource revealed a two-member NbFAD2 family, NbFAD2.1 and NbFAD2.2, and quantitative RT-PCR (qRT-PCR) confirmed their expression in leaves. FAD2 activities were silenced using hairpin RNAi as monitored by qRT-PCR and biochemical assays. Silencing of endogenous FAD2 activities was combined with overexpression of transgenes via the use of the alternative viral silencing-suppressor protein, V2, from Tomato yellow leaf curl virus. We show that V2 permits maximal overexpression of transgenes but, crucially, also allows hairpin RNAi to operate unimpeded. To illustrate the efficacy of the V2-based leaf assay system, endogenous lipids were shunted from the desaturation of 18:1 to elongation reactions beginning with 18:1 as substrate. These V2-based leaf assays produced ~50% more elongated fatty acid products than p19-based assays. Analyses of small RNA populations generated from hairpin RNAi against NbFAD2 confirm that the siRNA population is dominated by 21 and 22 nt species derived from the hairpin. Collectively, these new tools expand the range of uses and possibilities for metabolic engineering in transient leaf assays. © 2012 Naim et al.