Virion structure and in vitro genome release mechanism of dicistrovirus Kashmir bee virus.

Infections of Kashmir bee virus (KBV) are lethal for honeybees and have been associated with colony collapse disorder. KBV and closely related viruses contribute to the ongoing decline in the number of honeybee colonies in North America, Europe, Australia, and other parts of the world. Despite the economic and ecological impact of KBV, its structure and infection process remain unknown. Here we present the structure of the virion of KBV determined to a resolution of 2.8 Å. We show that the exposure of KBV to acidic pH induces a reduction in inter-pentamer contacts within capsids and the reorganization of its RNA genome from a uniform distribution to regions of high and low density. Capsids of KBV crack into pieces at acidic pH, resulting in the formation of open particles lacking pentamers of capsid proteins. The large openings of capsids enable the rapid release of genomes and thus limit the probability of their degradation by RNases. The opening of capsids may be a shared mechanism for the genome release of viruses from the family Dicistroviridae ImportanceThe western honeybee (Apis mellifera) is indispensable for maintaining agricultural productivity as well as the abundance and diversity of wild flowering plants. However, bees suffer from environmental pollution, parasites, and pathogens, including viruses. Outbreaks of virus infections cause the deaths of individual honeybees as well as collapses of whole colonies. Kashmir bee virus has been associated with colony collapse disorder in the US, and no cure of the disease is currently available. Here we report the structure of an infectious particle of Kashmir bee virus and show how its protein capsid opens to release the genome. Our structural characterization of the infection process determined that therapeutic compounds stabilizing contacts between pentamers of capsid proteins could prevent the genome release of the virus.

Dicistroviridae: A new viral purification technique.

The family Dicistroviridae comprises three genera and about twenty species of RNA virus, most of them with health or agricultural importance. The Triatoma virus (TrV) is the only entomopathogenic virus identified in triatomine bugs up to the present. TrV replicates within the intestinal epithelial cells, causing high mortality rate and delayed development of the molt of these bugs. TrV has been proposed as a biological control agent for vectors of Chagas disease. Viral particles were purified from feces of 1, 5 and 10 insects from an experimental colony of Triatoma infestans infected with TrV. Viral concentration and infectivity were corroborated using polyacrylamide gels and RT-PCR, respectively. In this work we report a method of viral purification that allows to reduce necessary reagents and time, using a very small amount of fecal matter.

X-ray structure of Triatoma virus empty capsid: insights into the mechanism of uncoating and RNA release in dicistroviruses.

In viruses, uncoating and RNA release are two key steps of successfully infecting a target cell. During these steps, the capsid must undergo the necessary conformational changes to allow RNA egress. Despite their importance, these processes are poorly understood in the family Dicistroviridae. Here, we used X-ray crystallography to solve the atomic structure of a Triatoma virus(TrV) empty particle (Protein Data Bank ID 5L7O), which is the resulting capsid after RNA release. It is observed that the overall shape of the capsid and of the three individual proteins is maintained in comparison with the mature virion. Furthermore, no channels indicative of RNA release are formed in the TrV empty particle. However, the most prominent change in the empty particle when compared with the mature virion is the loss of order in the N-terminal domain of the VP2 protein. In mature virions, the VP2 N-terminal domain of one pentamer is swapped with its twofold related copy in an adjacent pentamer, thereby stabilizing the binding between the pentamers. The loss of these interactions allows us to propose that RNA release may take place through transient flipping-out of pentameric subunits. The lower number of stabilizing interactions between the pentamers and the lack of formation of new holes support this model. This model differs from the currently accepted model for rhinoviruses and enteroviruses, in which genome externalization occurs by extrusion of the RNA through capsid channels.

Life History Traits and Demographic Parameters of Triatoma infestans (Hemiptera: Reduviidae) Fed on Human Blood.

Triatoma infestans (Klug, 1834) (Hemiptera: Reduviidae), the main vector of Chagas disease in South America, feeds primarily on humans, but ethical reasons preclude carrying out demographical studies using people. Thus, most laboratory studies of T. infestans are conducted using bird or mammal live hosts that may result in different demographic parameters from those obtained on human blood. Therefore, it is of interest to determine whether the use of an artificial feeder with human blood would be operational to rear triatomines and estimate population growth rates. We estimated life history traits and demographic parameters using an artificial feeder with human blood and compared them with those obtained on live hens. Both groups of T. infestans were kept under constant conditions [28 ± 1°C, 40 ± 5% relative humidity, a photoperiod of 12:12 (L:D) h] and fed weekly. On the basis of age-specific survival and age-specific fecundity, we calculated the intrinsic rate of natural increase (r), the finite rate of population growth (λ), the net reproductive rate (Ro), and the mean generation time (Tg). Our results show differences in life history traits between blood sources, resulting in smaller population growth rates on human blood than on live hens. Although demographic growth rate was smaller on human blood than on hens, it still remains positive, so the benefit/cost ratio of this feeding method seems relatively attractive. We discuss possibility of using the artificial feeder with human blood for both ecological and behavioral studies.

Structure of the Triatoma virus capsid.

The members of the Dicistroviridae family are non-enveloped positive-sense single-stranded RNA (+ssRNA) viruses pathogenic to beneficial arthropods as well as insect pests of medical importance. Triatoma virus (TrV), a member of this family, infects several species of triatomine insects (popularly named kissing bugs), which are vectors for human trypanosomiasis, more commonly known as Chagas disease. The potential use of dicistroviruses as biological control agents has drawn considerable attention in the past decade, and several viruses of this family have been identified, with their targets covering honey bees, aphids and field crickets, among others. Here, the crystal structure of the TrV capsid at 2.5 Å resolution is reported, showing that as expected it is very similar to that of Cricket paralysis virus (CrPV). Nevertheless, a number of distinguishing structural features support the introduction of a new genus (Triatovirus; type species TrV) under the Dicistroviridae family. The most striking differences are the absence of icosahedrally ordered VP4 within the infectious particle and the presence of prominent projections that surround the fivefold axis. Furthermore, the structure identifies a second putative autoproteolytic DDF motif in protein VP3, in addition to the conserved one in VP1 which is believed to be responsible for VP0 cleavage during capsid maturation. The potential meaning of these new findings is discussed.

American triatomine species occurrences: updates and novelties in the DataTri database.

The causative agent of Chagas disease (Trypanosoma cruzi) is transmitted to mammals, including humans, mainly by insect vectors of the subfamily Triatominae (Hemiptera: Reduviidae). Also known as "kissing bugs", the subfamily currently includes 157 validated species (154 extant and three extinct), in 18 genera and five tribes. Here, we present a subdataset (7852 records) of American triatomine occurrences; an update to the most complete and integrated database available to date at a continental scale. New georeferenced records were obtained from a systematic review of published literature and colleague-provided data. New data correspond to 101 species and 14 genera from 22 American countries between 1935 and 2022. The most important novelties refer to (i) the inclusion of new species, (ii) synonymies and formal transferals of species, and (iii) temporal and geographical species records updates. These data will be a useful contribution to entomological surveillance implicated in Chagas disease.

Publishing data to support the fight against human vector-borne diseases.

Vector-borne diseases are responsible for more than 17% of human cases of infectious diseases. In most situations, effective control of debilitating and deadly vector-bone diseases (VBDs), such as malaria, dengue, chikungunya, yellow fever, Zika and Chagas requires up-to-date, robust and comprehensive information on the presence, diversity, ecology, bionomics and geographic spread of the organisms that carry and transmit the infectious agents. Huge gaps exist in the information related to these vectors, creating an essential need for campaigns to mobilise and share data. The publication of data papers is an effective tool for overcoming this challenge. These peer-reviewed articles provide scholarly credit for researchers whose vital work of assembling and publishing well-described, properly-formatted datasets often fails to receive appropriate recognition. To address this, GigaScience's sister journal GigaByte partnered with the Global Biodiversity Information Facility (GBIF) to publish a series of data papers, with support from the Special Programme for Research and Training in Tropical Diseases (TDR), hosted by the World Health Organisation (WHO). Here we outline the initial results of this targeted approach to sharing data and describe its importance for controlling VBDs and improving public health.

Prevalence and distribution of parasites and pathogens of triatominae from Argentina, with emphasis on Triatoma infestans and Triatoma virus TrV.

Chagas' disease is the most important endemic arthropod-zoonosis in Argentina with an estimated 1.6 million people infected with the causative agent Trypanosoma cruzi. Triatoma infestans is the main vector of Chagas' disease in Argentina. A survey for parasites and pathogens of Triatominae was conducted from August 2002 to February 2005. Collections of insects were made in domiciles, peridomiciles, and in the natural habitats of the Triatominae. Insects from these collections were dissected and their organs and tissues examined for flagellates. Frass from these insects was collected and examined for detection of the entomopathogenic virus Triatoma virus (TrV) using AC-ELISA and PCR. Triatominae belonging to four species, T. infestans (n=1646), Triatoma guasayana (n=4), Triatoma platensis (n=1) and Triatoma sordida (n=5) were collected from 62 sites located in 13 provinces of Argentina. Triatoma virus and two protozoan species, Blastocrithidia triatomae and T. cruzi, the etiological agent of Chagas disease, were found infecting Triatominae. The total prevalence of TrV in 1646 T. infestans analyzed by ELISA was 9.66% (159/1646) from 7 to 13 provinces where collections were made. Triatoma virus positive triatomines were found in 17 of 62 populations when examined by AC-ELISA but in 38 of 62 populations when PCR was used for detection. The prevalence of B. triatomae in T. infestans was 0.43% (7/1646), while the prevalence of T. cruzi was 1.3% (21/1646). This is the first study on the diversity, distribution and prevalence of flagellated protozoa and TrV of Triatominae in endemic Chagas' disease regions of Argentina.

Prevalence of Harpellales from Chironomidae larvae in phytotelmata from punta Lara Forest, Argentina.

Harpellales (Zygomycota: Trichomycetes) fungi are cosmopolitan obligate inhabitants of the gut of immature insects. A biweekly survey of gut fungi associated with chironomid (Chironomidae: Diptera) larvae living in the impounded water from Eryngium cabrerae (Apiaceae) phytotelmata from Punta Lara forest, Argentina, was done Jan 2003-Dec 2004. Two species of Harpellales were associated with chironomid larvae, Smittium phytotelmatum in the hindgut of Polypedilum sp. and Stachylina lentica in the midgut of both Polypedilum sp. and Metriocnemus eryngiotelmatus. No statistically significant differences were recorded in the prevalence of these Harpellales between seasons. Environmental variables (temperature, rainfall and relative humidity), impounded water volume, pH and chironomid larval density did not have an effect on the prevalence of the Trichomycetes.

Exploring the role of genome and structural ions in preventing viral capsid collapse during dehydration.

Even though viruses evolve mainly in liquid milieu, their horizontal transmission routes often include episodes of dry environment. Along their life cycle, some insect viruses, such as viruses from the Dicistroviridae family, withstand dehydrated conditions with presently unknown consequences to their structural stability. Here, we use atomic force microscopy to monitor the structural changes of viral particles of Triatoma virus (TrV) after desiccation. Our results demonstrate that TrV capsids preserve their genome inside, conserving their height after exposure to dehydrating conditions, which is in stark contrast with other viruses that expel their genome when desiccated. Moreover, empty capsids (without genome) resulted in collapsed particles after desiccation. We also explored the role of structural ions in the dehydration process of the virions (capsid containing genome) by chelating the accessible cations from the external solvent milieu. We observed that ion suppression helps to keep the virus height upon desiccation. Our results show that under drying conditions, the genome of TrV prevents the capsid from collapsing during dehydration, while the structural ions are responsible for promoting solvent exchange through the virion wall.

DataTri, a database of American triatomine species occurrence.

Trypanosoma cruzi, the causative agent of Chagas disease, is transmitted to mammals - including humans - by insect vectors of the subfamily Triatominae. We present the results of a compilation of triatomine occurrence and complementary ecological data that represents the most complete, integrated and updated database (DataTri) available on triatomine species at a continental scale. This database was assembled by collecting the records of triatomine species published from 1904 to 2017, spanning all American countries with triatomine presence. A total of 21815 georeferenced records were obtained from published literature, personal fieldwork and data provided by colleagues. The data compiled includes 24 American countries, 14 genera and 135 species. From a taxonomic perspective, 67.33% of the records correspond to the genus Triatoma, 20.81% to Panstrongylus, 9.01% to Rhodnius and the remaining 2.85% are distributed among the other 11 triatomine genera. We encourage using DataTri information in various areas, especially to improve knowledge of the geographical distribution of triatomine species and its variations in time.

Seasonality and abundance of the mosquito Isostomyia paranensis from phytotelmata in temperate Argentina.

Sampling was conducted for 1 year in a marsh near Buenos Aires from axils of Scirpus giganteus, a larval habitat of the poorly known sabethine mosquito, Isostomyia paranensis. Immatures of this species were recovered on every sampling date, averaging 3-4/plant in April and decreasing to 0-1/plant in October-December. The spatial distribution of Is. paranensis immatures was clumped, and larval age skewed toward 1st instars. The percentage of mosquito-positive S. giganteus was negatively correlated with accumulated rainfall 1 wk before collection. Microcrustacea were the only other invertebrates common in this phytotelmata, and no parasites or pathogens were detected in Is. paranensis. Fourth instars of this species attacked and killed one another in the laboratory, but only algae were recovered from dissected digestive tracts of field-collected larvae. Adult females of this species emerged from independent collections of pupae refused blood, but females captured at human bait readily consumed human blood. Mean (+/- SD) number of eggs developed by females collected at human bait and fed with blood (77.4 +/- 22.8) was not significantly different from the mean number of eggs developed by females collected as pupae and fed on sugar (72.0 +/-23.0).

Predation efficiency of indigenous larvivorous fish species on Culex pipiens L. larvae (Diptera: Culicidae) in drainage ditches in Argentina.

Two neotropical freshwater fish species, Cnesterodom decemmaculatus (Poeciliidae) and Jenynsia multidentata (Anablepidae), were collected from human-made ditches, a common habitat of the house mosquito Culex pipiens in La Plata, Argentina. Cnesterodom decemmaculatus was recorded in 62 of the 100 examined ditches, whereas J. multidentata was collected from only 21 ditches sympatrically with C. decemmaculatus. Culex pipiens was the only mosquito species collected, and its larvae and pupae were found in 38 of the 100 ditches. Fish and mosquito larvae and pupae were collected together in only two ditches and were significantly negatively correlated. Siphons of larval Culex and remnants of chironomid larvae, copepods, aquatic mites, and fish were present in the gut contents of two C. decemmaculatus from mosquito-positive ditches, while diatoms and filamentous algae were recorded in every fish dissected. Adult C. decemmaculatus and J. multidentata needed approximately 6.2 h to completely digest one Cx. pipiens 4th instar larva under laboratory conditions. When fish were confined with a density of 60 or fewer Cx. pipiens 4th instar larvae, C. decemmaculatus and J. multidentata adults consumed 100% of them in one day but only 35% and 42%, respectively, when confined with 150 larvae. Eradication of Cx. pipiens from a ditch, where densities had averaged 250 immatures per dip, was achieved 17 days after the introduction of 1,700 C. decemmaculatus.

[Population dynamics of the immature stages of Aedes aegypti (Diptera: Culicidae), vector of dengue: a longitudinal study (1996-2000)]. / Dinámica poblacional de los estadios inmaduros del vector del dengue Aedes aegypti (Diptera: Culicidae): un estudio longitudinal (1996-2000).

A four year study was conducted on a natural population of immature stages of Aedes aegypti after the re-invasion of Argentina by this vector in 1987. Thirty six plastic containers with 700 ml of dechlorinated water were placed in the La Plata Zoological Garden, La Plata, Argentina. A strip of filter paper around each container was added to facilitate egg counting. Eggs, larvae and pupae were counted weekly in each container from September, 1996 to August, 2000. After egg counting, papers were submerged to facilitate egg hatching and a new paper was placed in each container. Presence of A. aegypti immature stages was recorded from December-January to June during each of the four years of this study. In 1997, 13,105 eggs, 7,978 larvae and 1,476 pupae were registered with 54.7 % positive containers; during 1998, 8,194 eggs, 668 larvae and 142 pupae were recorded with 28.3 % positive containers; 13,510 eggs, 3,690 larvae and 743 pupae were registered during 1999 with 56.7 % positive containers; and 16,327 eggs, 4,669 larvae and 715 pupae during 2000 with 59.3 % of containers with presence of A. aegypti. Egg number and hatching rate were drastically reduced in 1998 when temperatures from December to May were 1 to 2.5 degrees C lower than the other years of this study. These colder than usual temperatures in the summer of 1998 were a consequence of the El Niño event.

Triatomines intrahaemocoelic inoculation protocol: a useful tool to check infectivity in insects.

Vectors of Chagas disease are currently controlled by employing several chemical insecticides though there is a continuing search for alternative ecological methods against disease causing vectors. An effective method includes the use of specific pathogens as biological control agents. The aim of this work was to describe a complete experimental inoculation protocol in triatomines. The intrahaemocoelic inoculation technique can be applied to inoculate different kinds of microorganisms such as viruses, fungi, bacteria and protozoa; so it could be considered a useful tool in infective bioassays. This article includes results from evaluations of Triatoma virus (TrV, Dicistroviridae: Triatovirus) infectivity in several triatomine species. The protocol, also suitable for any other kind of insects, describes the materials and steps required to safely inoculate the insects, preventing any damage and/or contamination.

Trichomycetes living in the guts of aquatic insects of Misiones and Tierra del Fuego, Argentina.

Fourteen species of Trichomycetes living in the guts of aquatic insects are reported from two provinces of Argentina, Misiones and Tierra del Fuego. Twelve of the species belong to the Harpellales and two are Amoebidiales. Five harpellid species are reported from Misiones in the extreme northeast of the country (Genistellospora homothallica, Harpella tica, Smittium culisetae, Smittium sp., Stachylina sp.) and seven are from Tierra del Fuego, the southern tip of South America (H. meridianalis, Glotzia sp., S. culicis, S. cellaspora, S. imitatum, Stachylina minima, Penella simulii). Insect hosts all were immature stages of Culicidae, Simuliidae, Chironomidae, Ceratopogonidae (Insecta: Diptera), and Ephemeroptera and Plecoptera. The lower diversity of Trichomycetes found at Misiones, which has a subtropical climate and rainforest vegetation, was due possibly to the warmer temperatures of the water (15-24 C), compared to the colder streams of Tierra del Fuego (9-15 C), with forests and steppes as typical vegetation.

Seroprevalence of Triatoma virus (Dicistroviridae: Cripaviridae) antibodies in Chagas disease patients.

BACKGROUND: Chagas disease is caused by Trypanosoma cruzi, and humans acquire the parasite by exposure to contaminated feces from hematophagous insect vectors known as triatomines. Triatoma virus (TrV) is the sole viral pathogen of triatomines, and is transmitted among insects through the fecal-oral route and, as it happens with T. cruzi, the infected insects release the virus when defecating during or after blood uptake. METHODS: In this work, we analysed the occurrence of anti-TrV antibodies in human sera from Chagas disease endemic and non-endemic countries, and developed a mathematical model to estimate the transmission probability of TrV from insects to man, which ranged between 0.00053 and 0.0015. RESULTS: Our results confirm that people with Chagas disease living in Bolivia, Argentina and Mexico have been exposed to TrV, and that TrV is unable to replicate in human hosts. CONCLUSIONS: We presented the first experimental evidence of antibodies against TrV structural proteins in human sera.

Inoculation of Triatoma virus (Dicistroviridae: Cripavirus) elicits a non-infective immune response in mice.

BACKGROUND: Dicistroviridae is a new family of small, non-enveloped, +ssRNA viruses pathogenic to both beneficial arthropods and insect pests. Little is known about the dicistrovirus replication mechanism or gene function, and any knowledge on these subjects comes mainly from comparisons with mammalian viruses from the Picornaviridae family. Due to its peculiar genome organization and characteristics of the per os viral transmission route, dicistroviruses make good candidates for use as biopesticides. Triatoma virus (TrV) is a pathogen of Triatoma infestans (Hemiptera: Reduviidae), one of the main vectors of the human trypanosomiasis disease called Chagas disease. TrV was postulated as a potential control agent against Chagas' vectors. Although there is no evidence that TrV nor other dicistroviruses replicate in species outside the Insecta class, the innocuousness of these viruses in humans and animals needs to be ascertained. METHODS: In this study, RT-PCR and ELISA were used to detect the infectivity of this virus in Mus musculus BALB/c mice. RESULTS: In this study we have observed that there is no significant difference in the ratio IgG2a/IgG1 in sera from animals inoculated with TrV when compared with non-inoculated animals or mice inoculated only with non-infective TrV protein capsids. CONCLUSIONS: We conclude that, under our experimental conditions, TrV is unable to replicate in mice. This study constitutes the first test to evaluate the infectivity of a dicistrovirus in a vertebrate animal model.

Molecular techniques for dicistrovirus detection without RNA extraction or purification.

Dicistroviridae is a new family of small, nonenveloped, and +ssRNA viruses pathogenic to both beneficial arthropods and insect pests as well. Triatoma virus (TrV), a dicistrovirus, is a pathogen of Triatoma infestans (Hemiptera: Reduviidae), one of the main vectors of Chagas disease. In this work, we report a single-step method to identify TrV, a dicistrovirus, isolated from fecal samples of triatomines. The identification method proved to be quite sensitive, even without the extraction and purification of RNA virus.

Dicistroviridae: A new viral purification technique / Dicistroviridae: una nueva técnica de purificación viral

The family Dicistroviridae comprises three genera and about twenty species of RNA virus, most of them with health or agricultural importance. The Triatoma virus (TrV) is the only entomopathogenic virus identified in triatomine bugs up to the present. TrV replicates within the intestinal epithelial cells, causing high mortality rate and delayed development of the molt of these bugs. TrV has been proposed as a biological control agent for vectors of Chagas disease. Viral particles were purified from feces of 1, 5 and 10 insects from an experimental colony of Triatoma infestans infected with TrV. Viral concentration and infectivity were corroborated using polyacrylamide gels and RT-PCR, respectively. In this work we report a method of viral purification that allows to reduce necessary reagents and time, using a very small amount of fecal matter.

La familia Dicistroviridae está compuesta por tres géneros y casi una veintena de especies de virus ARN, la mayoría de ellas de importancia sanitaria o agrícola. Triatoma virus (TrV) es el único virus entomopatógeno identificado en triatominos hasta el momento. El TrV se replica en las células del epitelio intestinal; ello provoca una alta tasa de mortalidad y retraso en el desarrollo de la muda del insecto. Se ha propuesto la utilización de TrV como agente de control biológico para vectores de la enfermedad de Chagas. Las partículas virales fueron purificadas a partir de materia fecal de 1, 5 y 10 insectos obtenidos de una colonia experimental infectada con TrV de Triatoma infestans y se corroboró su concentración viral e infectividad mediante geles de poliacrilamida y RT-PCR, respectivamente. En este trabajo se reporta un método de purificación viral que permite la reducción de los reactivos y del tiempo necesario para lograr dicha purificación, partiendo de una mínima cantidad de materia fecal.