Transovarial transmission of a core virome in the Chagas disease vector Rhodnius prolixus.

Triatomine assassin bugs comprise hematophagous insect vectors of Trypanosoma cruzi, the causative agent of Chagas disease. Although the microbiome of these species has been investigated to some extent, only one virus infecting Triatoma infestans has been identified to date. Here, we describe for the first time seven (+) single-strand RNA viruses (RpV1-7) infecting Rhodnius prolixus, a primary vector of Chagas disease in Central and South America. We show that the RpVs belong to the Iflaviridae, Permutotetraviridae and Solemoviridae and are vertically transmitted from the mothers to the progeny via transovarial transmission. Consistent with this, all the RpVs, except RpV2 that is related to the entomopathogenic Slow bee paralysis virus, established persistent infections in our R. prolixus colony. Furthermore, we show that R. prolixus ovaries express 22-nucleotide viral siRNAs (vsiRNAs), but not viral piRNAs, that originate from the processing of dsRNA intermediates during viral replication of the RpVs. Interestingly, the permutotetraviruses and sobemoviruses display shared pools of vsiRNAs that might provide the basis for a cross-immunity system. The vsiRNAs are maternally deposited in the eggs, where they likely contribute to reduce the viral load and protect the developing embryos. Our results unveil for the first time a complex core virome in R. prolixus and begin to shed light on the RNAi-based antiviral defenses in triatomines.

A decade of vector control activities: Progress and limitations of Chagas disease prevention in a region of Guatemala with persistent Triatoma dimidiata infestation.

INTRODUCTION: Chagas disease, a neglected tropical disease that affects millions of Latin Americans, has been effectively controlled in Guatemala after multiple rounds of indoor residual insecticide spraying (IRS). However, a few foci remain with persistent Triatoma dimidiata infestation. One such area is the municipality of Comapa, Department of Jutiapa, in the southeastern region of Guatemala, where control interventions appear less effective. We carried out three cross sectional entomological and serological surveys in Comapa to evaluate a decade of vector control activities. Baseline serological (1999) and entomological (2001-2) surveys were followed by three rounds of insecticide applications (2003-2005) and intermittent focal spraying of infested houses, until approximately 2012. Household inspections to determine entomological indices and construction materials were conducted in 2001, 2007 and 2011. Seroprevalence surveys were conducted in school-age children in 1999, 2007 and 2015, and in women of child bearing age (15-44 years) only in 2015. After multiple rounds of indoor residual sprayings (IRS), the infestation index decreased significantly from 39% (2001-2) to 27% (2011). Household construction materials alone predicted <10% of infested houses. Chagas seroprevalence in Comapa declined in school-aged children by 10-fold, from 10% (1999) to 1% (2015). However, seroprevalence in women of child bearing age remains >10%. CONCLUSION: After a decade of vector control activities in Comapa, there is evidence of significantly reduced transmission. However, the continued risk for vector-borne and congenital transmission pose a threat to the 2022 Chagas disease elimination goal. Systematic integrated vector control and improved Chagas disease screening and treatment programs for congenital and vector-borne disease are needed to reach the elimination goal in regions with persistent vector infestation.

Can Triatoma virus inhibit infection of Trypanosoma cruzi (Chagas, 1909) in Triatoma infestans (Klug)? A cross infection and co-infection study.

Triatoma virus occurs infecting Triatominae in the wild (Argentina) and in insectaries (Brazil). Pathogenicity of Triatoma virus has been demonstrated in laboratory; accidental infections in insectaries produce high insect mortality. When more than one microorganism enters the same host, the biological interaction among them differs greatly depending on the nature and the infection order of the co-existing species of microorganisms. We studied the possible interactions between Triatoma virus (TrV) and Trypanosoma cruzi (the etiological agent of Chagas disease) in three different situations: (i) when Triatoma virus is inoculated into an insect host (Triatoma infestans) previously infected with T. cruzi, (ii) when T. cruzi is inoculated into T. infestans previously infected with TrV, and (iii) when TrV and T. cruzi are inoculated simultaneously into the same T. infestans individual. Trypanosoma cruzi infection was found in 57% of insects in the control group for T. cruzi, whereas 85% of insects with previous TrV infection were infected with T. cruzi. TrV infection was found in 78.7% of insects in the control group for TrV, whereas insects previously infected with T. cruzi showed 90% infection with TrV. A total of 67.9% of insects presented simultaneous infection with both types of microorganism. Our results suggest that TrV infection could increase adhesion of T. cruzi to the intestinal cells of triatomines, but presence of T. cruzi in intestinal cells would not increase the possibility of entry of TrV into cells. Although this study cannot explain the mechanism through which TrV facilitates the infection of triatomines with T. cruzi, we conclude that after TrV replication, changes at cellular level should occur that increase the adhesion of T. cruzi.

ICTV Virus Taxonomy Profile: Dicistroviridae.

Dicistroviridae is a family of small non-enveloped viruses with monopartite, linear, positive-sense RNA genomes of approximately 8-10 kb. Viruses of all classified species infect arthropod hosts, with some having devastating economic consequences, such as acute bee paralysis virus in domesticated honeybees and taura syndrome virus in shrimp farming. Conversely, the host specificity and other desirable traits exhibited by several members of this group make them potential natural enemies for intentional use against arthropod pests, such as triatoma virus against triatomine bugs that vector Chagas disease. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the taxonomy of the Dicistroviridae which is available at www.ictv.global/report/dicistroviridae.

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.

Modelling the potential geographic distribution of triatomines infected by Triatoma virus in the southern cone of South America.

BACKGROUND: Triatoma virus (TrV) is the only entomopathogenous virus identified in triatomines. We estimated the potential geographic distribution of triatomine species naturally infected by TrV, using remotely sensed and meteorological environmental variables, to predict new potential areas where triatomines infected with TrV may be found. METHODS: Detection of TrV infection in samples was performed with RT-PCR. Ecological niche models (ENM) were constructed using the MaxEnt software. We used 42 environmental variables derived from remotely sensed imagery (AVHRR) and 19 bioclimatic variables (Bioclim). The MaxEnt Jackknife procedure was used to minimize the number of environmental variables that showed an influence on final models. The goodness of fit of the model predictions was evaluated by the mean area under the curve (AUC). RESULTS: We obtained 37 samples of 7 species of triatomines naturally infected with TrV. Of the TrV positive samples, 32% were from sylvatic habitat, 46% came from peridomicile habitats and 22% from domicile habitats. Five of the seven infected species were found only in the sylvatic habitat, one species only in the domicile and only Triatoma infestans was found in the three habitats. The MaxEnt model estimated with the Bioclim dataset identified five environmental variables as best predictors: temperature annual range, mean diurnal range, mean temperature of coldest quarter, temperature seasonality and annual mean temperature. The model using the AVHRR dataset identified six environmental variables: minimum Land Surface Temperature (LST), minimum Middle Infrared Radiation (MIR), LST annual amplitude, MIR annual amplitude annual, LST variance and MIR variance. The potential geographic distribution of triatomine species infected by TrV coincides with the Chaco and the Monte ecoregions either modelled by AVHRR or Bioclim environmental datasets. CONCLUSIONS: Our results show that the conditions of the Dry Chaco ecoregion in Argentina are favourable for the infection of triatomine species with TrV, and open the possibility of its use as a potential agent for the biological control of peridomestic and/or sylvatic triatomine species. Results identify areas of potential occurrence that should be verified in the field.

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.

Detection of triatomine infection by Triatoma virus and horizontal transmission: protecting insectaries and prospects for biological control.

Triatoma virus (TrV) is the only triatomine entomopathogenic virus identified so far. Propagation of TrV in insectaries depends on handling procedures and triatomine population dynamics. The effects of propagation can be devastating and entire colonies must often be sacrificed to prevent spread of the virus throughout the insectary. This study found that after 41.3 days from TrV ingestion of human blood with 0.04 mg of viral protein by 5th instar Triatomainfestans, viral particles could be detected by RT-PCR; in a second horizontal transmission experiment time to detection resulted in a mean of 42.5 days. These results should rise awareness of TrV dynamics in nature, help estimate the spread of this virus when TrV-infected field-collected insects are incorporated into an insectary, and provide a base for the consideration of TrV as an agent of biological control of some species of triatomines.

First study of different insect cells to triatoma virus infection.

The use of viruses for biological control is a new option to be considered. The family Dicistroviridae, which affects only invertebrates, is one of the families that have been proposed for this purpose. The Triatoma virus (TrV), a member of this family, affects triatomine transmitters of Chagas disease, which is endemic in Latin America but also expanding its worldwide distribution. To this end, we attempted virus replication in Diptera, Aedes albopictus (clone C6/36) and Lepidoptera Spodoptera frugiperda (SF9, SF21) and High Five (H5) cell lines. The methodologies used were transfection process, direct inoculation (purified virus), and inoculation of purified virus with trypsin. Results were confirmed by SDS-PAGE, Western blotting, RT-PCR, electron microscopy, and immunofluorescence. According to the results obtained, further analysis of susceptibility/infection of H5 cells to TrV required to be studied.

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.

Probing the biophysical interplay between a viral genome and its capsid.

The interaction between a viral capsid and its genome governs crucial steps in the life cycle of a virus, such as assembly and genome uncoating. Tuning cargo-capsid interactions is also essential for successful design and cargo delivery in engineered viral systems. Here we investigate the interplay between cargo and capsid for the picorna-like Triatoma virus using a combined native mass spectrometry and atomic force microscopy approach. We propose a topology and assembly model in which heterotrimeric pentons that consist of five copies of structural proteins VP1, VP2 and VP3 are the free principal units of assembly. The interpenton contacts are established primarily by VP2. The dual role of the genome is first to stabilize the densely packed virion and, second, on an increase in pH to trigger uncoating by relaxing the stabilizing interactions with the capsid. Uncoating occurs through a labile intermediate state of the virion that reversibly disassembles into pentons with the concomitant release of protein VP4.

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.

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.

Cryo-electron microscopy reconstructions of triatoma virus particles: a clue to unravel genome delivery and capsid disassembly.

Triatoma virus (TrV) is a member of the insect virus family Dicistroviridae and consists of a small, non-enveloped capsid that encloses its positive-sense ssRNA genome. Using cryo-transmission electron microscopy and three-dimensional reconstruction techniques combined with fitting of the available crystallographic models, this study analysed the capsids corresponding to mature and several RNA-empty TrV particles. After genome release, the resulting reconstruction of the empty capsids displayed no prominent conformational changes with respect to the full virion capsid. The results showed that RNA delivery led to empty capsids with an apparent overall intact protein shell and suggested that, in a subsequent step, empty capsids disassemble into small symmetrical particles. Contrary to what is observed upon genome release in mammalian picornaviruses, the empty TrV capsid maintained a protein shell thickness and size identical to that in full virions.

New Triatoma virus hosts in wild habitats of Argentina.

Triatoma virus (TrV), a member of the Dicistroviridae family, replicates in intestinal epithelial cells, causing delayed development and death of infected individuals. The aims of this study were to find naturally infected species of Triatominae in the wild in the region endemic for Chagas disease and analyze and compare the sequence diversity of TrV obtained from different Triatominae. A total of 253 Triatominae belonging to 10 species were captured by active or passive collection. Three new sequences were obtained from Triatoma infestans, Triatoma delpontei and Psammolestes coreodes and the analysis revealed that these sequences were very similar. Ps. coreodes is a new host for TrV.

Capsid protein identification and analysis of mature Triatoma virus (TrV) virions and naturally occurring empty particles.

Triatoma virus (TrV) is a non-enveloped +ssRNA virus belonging to the insect virus family Dicistroviridae. Mass spectrometry (MS) and gel electrophoresis were used to detect the previously elusive capsid protein VP4. Its cleavage sites were established by sequencing the N-terminus of the protein precursor and MS, and its stoichiometry with respect to the other major capsid proteins (VP1-3) was found to be 1:1. We also characterized the polypeptides comprising the naturally occurring non-infectious empty capsids, i.e., RNA-free TrV particles. The empty particles were composed of VP0-VP3 plus at least seven additional polypeptides, which were identified as products of the capsid precursor polyprotein. We conclude that VP4 protein appears as a product of RNA encapsidation, and that defective processing of capsid proteins precludes genome encapsidation.

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.

AC-ELISA and RT-PCR assays for the diagnosis of triatoma virus (TrV) in triatomines (Hemiptera: Reduviidae) species.

Triatoma virus (TrV) is the only entomopathogenic virus found in triatomines. TrV replicates in cells of the midgut epithelium of triatomines, causing a high mortality rate and delayed development of the infected insect. In this work, we report an antigen-capture enzyme-linked immunosorbent assay (AC-ELISA) and a reverse transcription-polymerase chain reaction (RT-PCR) assay for detection of TrV infection. For antiserum production, rabbits and hens where inoculated with purified TrV. Antiserum reactivity was checked by immunodiffusion, and its specificity was confirmed by western blot and AC-ELISA. Totally 90 fecal samples from T. infestans were analysed. AC-ELISA and RT-PCR results correlated well with transmission electron microscopy (EM) observations, which are considered the gold standard, with Kappa values of 0.73 for AC-ELISA and 0.93 for RT-PCR when compared with EM. Applications and complementary uses of the two techniques reported in this work are discussed.

Phasing of the Triatoma virus diffraction data using a cryo-electron microscopy reconstruction.

The blood-sucking reduviid bug Triatoma infestans, one of the most important vector of American human trypanosomiasis (Chagas disease) is infected by the Triatoma virus (TrV). TrV has been classified as a member of the Cripavirus genus (type cricket paralysis virus) in the Dicistroviridae family. This work presents the three-dimensional cryo-electron microscopy (cryo-EM) reconstruction of the TrV capsid at about 25 A resolution and its use as a template for phasing the available crystallographic data by the molecular replacement method. The main structural differences between the cryo-EM reconstruction of TrV and other two viruses, one from the same family, the cricket paralysis virus (CrPV) and the human rhinovirus 16 from the Picornaviridae family are presented and discussed.

Characterization of internal ribosomal entry sites of Triatoma virus.

Triatoma virus (TrV) belongs to a new family of RNA viruses known as Dicistroviridae. Nucleotide sequence comparisons between different dicistroviruses allowed two putative internal ribosomal entry sites (IRESs) in the TrV RNA to be defined: the 5'UTR IRES of 548 nt and the intergenic region (IGR) IRES of 172 nt. Using monocistronic and bicistronic RNAs, it was shown that the TrV genome contains two functional IRESs that mediate translation initiation in a cap-independent manner. In addition, it was found that the two TrV IRESs were able to direct efficient translation of reporter genes in microinjected Xenopus oocytes, suggesting minimum requirements for host factors. The IGR IRES begins with a non-canonical CUC; however, mutations of this triplet to AUG or CCU did not impair IRES function, indicating that the CUC is not essential for the initiation process. Furthermore, translation efficiency from two TrV IRESs was differentially modulated by IFN-alpha and viral infection.