Transcriptome markers of viral persistence in naturally-infected andes virus (bunyaviridae) seropositive long-tailed pygmy rice rats.

Long-tailed pygmy rice rats (Oligoryzomys longicaudatus) are principal reservoir hosts of Andes virus (ANDV) (Bunyaviridae), which causes most hantavirus cardiopulmonary syndrome cases in the Americas. To develop tools for the study of the ANDV-host interactions, we used RNA-Seq to generate a de novo transcriptome assembly. Splenic RNA from five rice rats captured in Chile, three of which were ANDV-infected, was used to generate an assembly of 66,173 annotated transcripts, including noncoding RNAs. Phylogenetic analysis of selected predicted proteins showed similarities to those of the North American deer mouse (Peromyscus maniculatus), the principal reservoir of Sin Nombre virus (SNV). One of the infected rice rats had about 50-fold more viral burden than the others, suggesting acute infection, whereas the remaining two had levels consistent with persistence. Differential expression analysis revealed distinct signatures among the infected rodents. The differences could be due to 1) variations in viral load, 2) dimorphic or reproductive differences in splenic homing of immune cells, or 3) factors of unknown etiology. In the two persistently infected rice rats, suppression of the JAK-STAT pathway at Stat5b and Ccnot1, elevation of Casp1, RIG-I pathway factors Ppp1cc and Mff, and increased FC receptor-like transcripts occurred. Caspase-1 and Stat5b activation pathways have been shown to stimulate T helper follicular cell (TFH) development in other species. These data are also consistent with reports suggestive of TFH stimulation in deer mice experimentally infected with hantaviruses. In the remaining acutely infected rice rat, the apoptotic pathway marker Cox6a1 was elevated, and putative anti-viral factors Abcb1a, Fam46c, Spp1, Rxra, Rxrb, Trmp2 and Trim58 were modulated. Transcripts for preproenkephalin (Prenk) were reduced, which may be predictive of an increased T cell activation threshold. Taken together, this transcriptome dataset will permit rigorous examination of rice rat-ANDV interactions and may lead to better understanding of virus ecology.

Rapid enzyme-linked immunosorbent assay for the detection of hantavirus-specific antibodies in divergent small mammals.

We assessed the utility of an enzyme-linked immunosorbent assay (ELISA) for the detection of hantavirus-specific antibodies from sera of Oligoryzomys longicaudatus, the principal reservoir of Andes virus (ANDV), using an antigen previously developed for detection of antibodies to Sin Nombre virus (SNV) in sera from Peromyscus maniculatus. The assay uses a protein A/G horseradish peroxidase conjugate and can be performed in as little as 1.5 hours. Serum samples from Oligoryzomys longicaudatus collected in central-south Chile were used and the assay identified several that were antibody positive. This assay can be used for the rapid detection of antibodies to divergent hantaviruses from geographically and phylogenetically distant rodent species.

Kinetics of immune responses in deer mice experimentally infected with Sin Nombre virus.

Deer mice are the principal reservoir hosts of Sin Nombre virus, the etiologic agent of most hantavirus cardiopulmonary syndrome cases in North America. Infection of deer mice results in persistence without conspicuous pathology, and most, if not all, infected mice remain infected for life, with periods of viral shedding. The kinetics of viral load, histopathology, virus distribution, and immune gene expression in deer mice were examined. Viral antigen was detected as early as 5 days postinfection and peaked on day 15 in the lungs, hearts, kidneys, and livers. Viral RNA levels varied substantially but peaked on day 15 in the lungs and heart, and antinucleocapsid IgG antibodies appeared in some animals on day 10, but a strong neutralizing antibody response failed to develop during the 20-day experiment. No clinical signs of disease were observed in any of the infected deer mice. Most genes were repressed on day 2, suggesting a typical early downregulation of gene expression often observed in viral infections. Several chemokine and cytokine genes were elevated, and markers of a T cell response occurred but then declined days later. Splenic transforming growth factor beta (TGF-ß) expression was elevated early in infection, declined, and then was elevated again late in infection. Together, these data suggest that a subtle immune response that fails to clear the virus occurs in deer mice.

Differential pattern of infection of sylvatic nymphs and domiciliary adults of Triatoma infestans with Trypanosoma cruzi genotypes in Chile.

In Chile, the main vector of Chagas disease, Triatoma infestans, is under control after insecticide spraying. However, it has been found colonizing wild habitats. This study evaluated Trypanosoma cruzi infection of sylvatic and domiciliary T. infestans and identified their parasite genotypes. The sample studied was composed mainly of T. infestans sylvatic nymphs and domiciliary adults from a semi-urban area with human dwellings under vector control surveillance. Results showed prevalences of 57.7% in nymphs and 68.6% in adults. Hybridization tests showed a major T. cruzi lineage (TcI) circulating in sylvatic (93.3%) and domiciliary (100%) T. infestans. TcII, TcV, and TcVI were also detected, mainly in nymphs, suggesting differential adaptation of T. cruzi lineages among instars. We also discuss the origin of domiciliary individuals of T. infestans and the risk of human infection by triatomines of sylvatic foci that invade houses despite vector control programs.

Statistical phylogeography of Chagas disease vector Triatoma infestans: testing biogeographic hypotheses of dispersal.

Chagas disease is one of the most important vector-borne diseases in Latin America. The disease, caused by the flagellate protozoan Trypanosoma cruzi, is commonly transmitted to humans by Triatoma infestans in South America. Using mitochondrial DNA sequences, we assessed alternative biogeographic scenarios of dispersal of T. infestans using coalescence simulations. We also assessed phylogeographic structure and spatial genetics of T. infestans in Chile. Two major routes of dispersal in southern South America were supported including a dual-origin of T. infestans in Chile. Phylogeographic analyses identified two primary clades with Chilean haplotypes partitioned into either a northern cluster with Peruvian and Bolivian haplotypes or a north-central cluster with Argentinean and Uruguayan haplotypes. The north-central clade is further divided into two subgroups. Domestic and sylvatic T. infestans in central Chile were not segregated in the phylogeographic reconstruction. Spatial genetic analyses show higher distances in northern Chile, congruent with the presence of two divergent lineages of T. infestans. Phylogenetic evidence does not unequivocally support the hypothesized Bolivian origin of T. infestans, so we discuss alternative scenarios.

New World hantaviruses activate IFNlambda production in type I IFN-deficient vero E6 cells.

BACKGROUND: Hantaviruses indigenous to the New World are the etiologic agents of hantavirus cardiopulmonary syndrome (HCPS). These viruses induce a strong interferon-stimulated gene (ISG) response in human endothelial cells. African green monkey-derived Vero E6 cells are used to propagate hantaviruses as well as many other viruses. The utility of the Vero E6 cell line for virus production is thought to owe to their lack of genes encoding type I interferons (IFN), rendering them unable to mount an efficient innate immune response to virus infection. Interferon lambda, a more recently characterized type III IFN, is transcriptionally controlled much like the type I IFNs, and activates the innate immune system in a similar manner. METHODOLOGY/PRINCIPAL FINDINGS: We show that Vero E6 cells respond to hantavirus infection by secreting abundant IFNlambda. Three New World hantaviruses were similarly able to induce IFNlambda expression in this cell line. The IFNlambda contained within virus preparations generated with Vero E6 cells independently activates ISGs when used to infect several non-endothelial cell lines, whereas innate immune responses by endothelial cells are specifically due to viral infection. We show further that Sin Nombre virus replicates to high titer in human hepatoma cells (Huh7) without inducing ISGs. CONCLUSIONS/SIGNIFICANCE: Herein we report that Vero E6 cells respond to viral infection with a highly active antiviral response, including secretion of abundant IFNlambda. This cytokine is biologically active, and when contained within viral preparations and presented to human epithelioid cell lines, results in the robust activation of innate immune responses. We also show that both Huh7 and A549 cell lines do not respond to hantavirus infection, confirming that the cytoplasmic RNA helicase pathways possessed by these cells are not involved in hantavirus recognition. We demonstrate that Vero E6 actively respond to virus infection and inhibiting IFNlambda production in these cells might increase their utility for virus propagation.

Temporal variation of Trypanosoma cruzi infection in native mammals in Chile.

In the present study, we compared Trypanosoma cruzi infection in four native mammals from a hyperendemic area of Chagas disease in Chile for two different periods to assess the occurrence of interannual variation (1999-2000 vs. 2005-2006). Parasite detection in mammals is performed by polymerase chain reaction assays and confirmed by Southern blot analysis and hybridization test with a universal probe. Results showed significant differences in the levels of T. cruzi infection between the compared periods. We suggest that the major El Niño event occurred in 1997-1998, a large-scale global climatic fluctuation, could be indirectly explaining the extremely high T. cruzi infection in 1999-2000 by means of a time-lag response of the wild transmission cycle of Chagas disease in semiarid Chile after the irruption of small rodent populations.

European rabbits (Oryctolagus cuniculus) are naturally infected with different Trypanosoma cruzi genotypes.

Trypanosoma cruzi, the etiologic agent of Chagas disease, presents a complex life cycle, cycling between reduviid vectors and wild and domestic mammals. The European rabbit is an introduced species in America, but its role as reservoir in the wild transmission cycle of Chagas disease remains unknown. We used polymerase chain reaction, Southern blot, and hybridization tests to detect infection and characterize genotypes in rabbits from a hyperendemic area of Chagas disease in Chile. Results show 38% of infection with different genotypes. We provide evidence that rabbits are naturally infected with T. cruzi, which may have important epidemiologic consequences for the wild transmission cycle.

Trypanosoma cruzi infection in the sylvatic kissing bug Mepraia gajardoi from the Chilean Southern Pacific Ocean coast.

The Southern Pacific Ocean coast has been traditionally considered a non-active transmission area for Chagas disease. In this report, we show evidence of Trypanosoma cruzi infection in the sylvatic kissing bug Mepraia gajardoi from the northern Chilean coast.

Conducta de picada y defecación de Mepraia spinolai en dos hospederos frecuentes en su habitat / Feedind behaviour of Mepraia spinolai in two frequent hosts from its habitat

The aim of this study was to compare the feeding and defecation behavior of wild and laboratory-reared specimens of Mepraia spinolai confronted to the rabbit, Oryctolagus cuniculus, and the wild rodent, Octodon degus as hosts. Time variations in bite, blood ingestion and defecation were analized, including weight gain after a blood meal. Considering bite duration significant differences were found according to the origin of insect. Laboratory reared specimens spent less biting time when fed on rabbit. No differences were found in blood ingestion time nor in defecation time. Blood meal was significantly different according to origin of the insect. Wild specimens of M. spinolaifed more blood than the laboratory ones. From these results it was apparent that there are not significant differences in the bug behavior when they are confronted to these hosts, being both mammals of a similar epidemiological importance.

Se estudió la conducta de alimentación y defecación de ejemplares de Mepraia spinolai provenientes de laboratorio y terreno alimentados sobre conejo, Oryctolagus cuniculus, y sobre roedores degu, Octodon degus. Se analizaron las diferencias en el tiempo de picada (latencia de picada), tiempo de ingesta de sangre, tiempo de defecación y cantidad de sangre ingerida en condiciones de laboratorio. El análisis del tiempo de picada, mostró que hubo diferencias significativas según origen de las vinchucas; esto es que los ejemplares de laboratorio demoraron el menor tiempo en picar a los conejos de terreno con el mismo hospedero, quienes tomaron el mayor tiempo. No hubo diferencias significativas para el tiempo de ingesta de sangre ni para el tiempo de defecación. Se encontraron diferencias entre grupos al comparar la cantidad de sangre ingerida; los insectos provenientes del terreno consumieron la mayor proporción de sangre comparados con los criados en laboratorio. De acuerdo con los resultados, estos insectos son generalistas en cuanto a hospederos y por tanto ninguno de ellos demostró tener una mayor importancia en el rol epidemiológico de mantención del parásito en el ciclo silvestre de la enfermedad.

Susceptibility of Mepraia spinolai and Triatoma infestans to different Trypanosoma cruzi strains from naturally infected rodent hosts.

Trypanosoma cruzi is the causative agent of Chagas disease, a zoonosis involving domestic and sylvatic mammalian reservoirs. Since scarce information has been published about the susceptibility of T. cruzi lineages to other triatomine species besides Triatoma infestans, we evaluate the susceptibility of T. infestans and Mepraia spinolai to different T. cruzi lineages, originated from naturally infected Octodon degus rodents as mammal host. Xenodiagnosis-PCR methods to detect T. cruzi positive rodents and genotyping to differentiate T. cruzi lineages (TcI, TcIIb, TcIId and TcIIe) identified singly and mixed T. cruzi infections. More infections and nearly all mixed infections were identified using the wild vector M. spinolai than T. infestans.