Deep Sequencing to Reveal Phylo-Geographic Relationships of Juquitiba Virus in Paraguay.

Several hantaviruses result in zoonotic infections of significant public health concern, causing hemorrhagic fever with renal syndrome (HFRS) or hantavirus cardiopulmonary syndrome (HCPS) in the Old and New World, respectively. Given a 35% case fatality rate, disease-causing New World hantaviruses require a greater understanding of their biology, genetic diversity, and geographical distribution. Juquitiba hantaviruses have been identified in Oligoryzomys nigripes in Brazil, Paraguay, and Uruguay. Brazil has reported the most HCPS cases associated with this virus. We used a multiplexed, amplicon-based PCR strategy to screen and deep-sequence the virus harbored within lung tissues collected from Oligoryzomys species during rodent field collections in southern (Itapúa) and western (Boquerón) Paraguay. No Juquitiba-like hantaviruses were identified in Boquerón. Herein, we report the full-length S and M segments of the Juquitiba hantaviruses identified in Paraguay from O. nigripes. We also report the phylogenetic relationships of the Juquitiba hantaviruses in rodents collected from Itapúa with those previously collected in Canindeyú. We showed, using the TN93 nucleotide substitution model, the coalescent (constant-size) population tree model, and Bayesian inference implemented in the Bayesian evolutionary analysis by sampling trees (BEAST) framework, that the Juquitiba virus lineage in Itapúa is distinct from that in Canindeyú. Our spatiotemporal analysis showed significantly different time to the most recent ancestor (TMRA) estimates between the M and S segments, but a common geographic origin. Our estimates suggest the additional geographic diversity of the Juquitiba virus within the Interior Atlantic Forest and highlight the need for more extensive sampling across this biome.

Deep spatial profiling of Venezuelan equine encephalitis virus reveals increased genetic diversity amidst neuroinflammation and cell death during brain infection.

Venezuelan equine encephalitis virus (VEEV) causes a febrile illness that can progress to neurological disease with the possibility of death in human cases. The evaluation and optimization of therapeutics that target brain infections demands knowledge of the host's response to VEEV, the dynamics of infection, and the potential for within-host evolution of the virus. We hypothesized that selective pressures during infection of the brain may differ temporally and spatially and so we investigated the dynamics of the host response, viral transcript levels, and genetic variation of VEEV TC-83 in eight areas of the brain in mice over 7 days post-infection (dpi). Viral replication increased throughout the brain until 5-6 dpi and decreased thereafter with neurons as the main site of viral replication. Low levels of genetic diversity were noted on 1 dpi and were followed by an expansion in the genetic diversity of VEEV and nonsynonymous (Ns) mutations that peaked by 5 dpi. The pro-inflammatory response and the influx of immune cells mirrored the levels of virus and correlated with substantial damage to neurons by 5 dpi and increased activation of microglial cells and astrocytes. The prevalence and dynamics of Ns mutations suggest that the VEEV is under selection within the brain and that progressive neuroinflammation may play a role in acting as a selective pressure. IMPORTANCE Treatment of encephalitis in humans caused by Venezuelan equine encephalitis virus (VEEV) from natural or aerosol exposure is not available, and hence, there is a great interest to address this gap. In contrast to natural infections, therapeutic treatment of infections from aerosol exposure will require fast-acting drugs that rapidly penetrate the blood-brain barrier, engage sites of infection in the brain and mitigate the emergence of drug resistance. Therefore, it is important to understand not only VEEV pathogenesis, but the trafficking of the viral population within the brain, the potential for within-host evolution of the virus, and how VEEV might evolve resistance.

Efficacy of a brain-penetrant antiviral in lethal Venezuelan and eastern equine encephalitis mouse models.

Venezuelan and eastern equine encephalitis viruses (VEEV and EEEV, respectively) are mosquito-borne, neuroinvasive human pathogens for which no FDA-approved therapeutic exists. Besides the biothreat posed by these viruses when aerosolized, arthropod transmission presents serious health risks to humans, as demonstrated by the 2019 outbreak of EEE disease in the United States that resulted in 38 confirmed cases, 19 deaths, and neurological effects in survivors. Here, we describe the discovery of a 2-pyrrolidinoquinazolinone scaffold, efficiently synthesized in two to five steps, whose structural optimization resulted in profound antiviral activity. The lead quinazolinone, BDGR-49, potently reduced cellular VEEV and EEEV titers by >7 log at 1 µM and exhibited suitable intravenous and oral pharmacokinetic profiles in BALB/c mice to achieve excellent brain exposure. Outstanding in vivo efficacy was observed in several lethal, subcutaneous infection mouse models using an 8-day dosing regimen. Prophylactically administered BDGR-49 at 25 mg kg-1 per day fully protected against a 10× LD50 VEEV Trinidad donkey (TrD) challenge in BALB/c mice. Similarly, we observed 70% protection when 10× LD50 EEEV FL93-939-infected C57BL/6 mice were treated prophylactically with BDGR-49 at 50 mg kg-1 per day. Last, we observed 100% therapeutic efficacy when mice, challenged with 10× LD50 VEEV TrD, were dosed at 48 hours after infection with BDGR-49 at 25 mg kg-1 per day. Mouse brain viral titers at 96 hours after infection were reduced to values near the limit of detection. Collectively, these results underscore the substantial development potential of a well-tolerated, brain-penetrant lead compound that shows promise in preventing and treating encephalitic alphavirus disease.

Screening of febrile patients with suspected malaria from the Brazilian Amazon for virus infection.

Arthropod-borne viruses (arboviruses) are a significant public health threat, especially in tropical and subtropical regions. More than 150 arboviruses can cause febrile illness following infection in humans. The Brazilian Amazon region has the highest number of arboviruses detected worldwide. In addition to arboviruses, malaria, caused by Plasmodium vivax, is endemic in the Amazon. Patients with malaria and arboviral disease frequently show similar clinical presentation and laboratory findings, making the diagnosis of the cause of the infection challenging. The aim of this study was to evaluate the potential for viral infections in patients with suspected malaria but without Plasmodium infection in the Brazilian Amazon. We recruited 200 subjects with suspected malaria in Manaus, Brazil. First, we tested for arboviruses in serum samples from 124 of the 200 participants using an arbovirus DNA microarray platform, which did not detect any virus. Then, we mixed the serum samples of the other 76 participants in 10 pools and subjected them to next-generation sequencing. Analysis of the sequencing data revealed the presence of only one arbovirus (Zika virus) in one sample pool. This analysis also detected the presence of primate erythroparvovirus 1 and pegivirus C. These results suggest that arboviruses are not the most frequent viral infections in patients with suspected malaria but without Plasmodium infection in the metropolitan region of Manaus. Implementation of specific viral surveillance tests will help in the early detection of viruses with epidemic potential.

Emergence and Magnitude of ML336 Resistance in Venezuelan Equine Encephalitis Virus Depend on the Microenvironment.

Venezuelan equine encephalitis virus (VEEV) is a New World Alphavirus that can cause neurological disease and death in humans and equines following transmission from infected mosquitoes. Despite the continued epidemic threat of VEEV, and its potential use as a bioterrorism agent, there are no FDA-approved antivirals or vaccines for treatment or prevention. Previously, we reported the discovery of a small molecule, ML336, with potent antiviral activity against VEEV. To further explore the population-level resistance profiles of ML336, we developed a whole-genome next-generation sequencing (NGS) approach to examine single nucleotide polymorphisms (SNPs) from virus passaged in dose escalation studies in a nonhuman primate kidney epithelial and a human astrocyte cell line, Vero 76 and SVGA, respectively. We passaged VEEV TC-83 in these two cell lines over seven concentrations of ML336, starting at 50 nM. NGS revealed several prominent mutations in the nonstructural protein (nsP) 3 and nsP4 genes that emerged consistently in these two distinct in vitro environments-notably, a mutation at Q210 in nsP4. Several of these mutations were stable following passaging in the absence of ML336 in Vero 76 cells. Network analyses showed that the trajectory of resistance differed between Vero and SVGA. Moreover, the penetration of SNPs was lower in SVGA. In conclusion, we show that the microenvironment influenced the SNP profile of VEEV TC-83. Understanding the dynamics of resistance in VEEV against newly developed antiviral compounds will guide the design of optimal drug candidates and dosing regimens for minimizing the emergence of resistant viruses.IMPORTANCE RNA viruses, including Venezuelan equine encephalitis virus (VEEV), have high mutation rates that allow for rapid adaptation to selective pressures in their environment. Antiviral compounds exert one such pressure on virus populations during infections. Next-generation sequencing allows for examination of viruses at the population level, which enables tracking of low levels of single-nucleotide polymorphisms in the population over time. Therefore, the timing and extent of the emergence of resistance to antivirals can be tracked and assessed. We show here that in VEEV, the trajectory and penetration of antiviral resistance reflected the microenvironment in which the virus population replicates. In summary, we show the diversity of VEEV within a single population under antiviral pressure and two distinct cell types, and we show that population dynamics in these viruses can be examined to better understand how they evolve over time.

A new inactivation method to facilitate cryo-EM of enveloped, RNA viruses requiring high containment: A case study using Venezuelan Equine Encephalitis Virus (VEEV).

The challenges associated with operating electron microscopes (EM) in biosafety level 3 and 4 containment facilities have slowed progress of cryo-EM studies of high consequence viruses. We address this gap in a case study of Venezuelan Equine Encephalitis Virus (VEEV) strain TC-83. Chemical inactivation of viruses may physically distort structure, and hence to verify retention of native structure, we selected VEEV strain TC-83 to develop this methodology as this virus has a 4.8 Šresolution cryo-EM structure. In our method, amplified VEEV TC-83 was concentrated directly from supernatant through a 30 % sucrose cushion, resuspended, and chemically inactivated with 1 % glutaraldehyde. A second 30 % sucrose cushion removed any excess glutaraldehyde that might interfere with single particle analyses. A cryo-EM map of fixed, inactivated VEEV was determined to a resolution of 7.9 Å. The map retained structural features of the native virus such as the icosahedral symmetry, and the organization of the capsid core and the trimeric spikes. Our results suggest that our strategy can easily be adapted for inactivation of other enveloped, RNA viruses requiring BSL-3 or BSL-4 for cryo-EM. However, the validation of inactivation requires the oversight of Biosafety Committee for each Institution.

Benzamidine ML336 inhibits plus and minus strand RNA synthesis of Venezuelan equine encephalitis virus without affecting host RNA production.

Venezuelan equine encephalitis virus (VEEV) is an alphavirus that is endemic to the Americas. VEEV outbreaks occur periodically and cause encephalitis in both humans and equids. There are currently no therapeutics or vaccines for treatment of VEEV in humans. Our group has previously reported on the development of a benzamidine VEEV inhibitor, ML336, which shows potent antiviral activity in both in vitro and in vivo models of infection. In cell culture experiments, ML336 inhibits viral RNA synthesis when added 2-4 h post-infection, and mutations conferring resistance occur within the viral nonstructural proteins (nsP2 and nsP4). We hypothesized that ML336 targets an activity of the viral replicase complex and inhibits viral RNA synthesis. To test this hypothesis, we employed various biochemical and cellular assays. Using structural analogues of ML336, we demonstrate that the cellular antiviral activity of these compounds correlates with their inhibition of viral RNA synthesis. For instance, the IC50 of ML336 for VEEV RNA synthesis inhibition was determined as 1.1 nM, indicating potent anti-RNA synthesis activity in the low nanomolar range. While ML336 efficiently inhibited VEEV RNA synthesis, a much weaker effect was observed against the Old World alphavirus Chikungunya virus (IC50 > 4 µM), agreeing with previous data from a cell based assay. Using a tritium incorporation assay, we demonstrated that there was no significant inhibition of cellular transcription. With a combination of fluorography, strand-specific qRT-PCR, and tritium incorporation, we demonstrated that ML336 inhibits the synthesis of the positive sense genomic, negative sense template, and subgenomic RNAs of VEEV. Based on these results, we propose that the mechanism of action for this class of antiviral compounds is inhibition of viral RNA synthesis through interaction with the viral replicase complex.

Sympatry and habitat associations of sigmodontine rodents in a neotropical forest-savanna interface.

Small mammal communities in the Neotropics are composed largely of sigmodontine rodents. However, many questions regarding these communities remain unanswered, especially those pertaining to fine-scale sympatry and habitat selection. To address this, we examined sigmodontine community structure and vegetation in the western margin of the Upper Paraná Atlantic Forest and the southwestern-most extent of the Cerrado (CE) (an extensive South American savanna ecoregion) of Paraguay. Vegetation classifications were derived from satellite imagery combined with maps based on extensive ground-based surveys. The three most abundant species (Akodon montensis, Hylaeamys megacephalus, and Oligoryzomys nigripes) were found most often in microsympatry with conspecifics, and were negatively associated with other species. Akodon montensis was associated with high forest (HF), and H. megacephalus with bamboo understory (BU), whereas O. nigripes did not exhibit a habitat preference. The first two species' distributions within the landscape were found to be driven primarily by habitat selection, and O. nigripes by a behavioral response (avoidance) to the presence of the other two species. Moreover, habitat influences whether or not a particular species associates with, or avoids, conspecifics or other species.

Efficacy of a ML336 derivative against Venezuelan and eastern equine encephalitis viruses.

Currently, there are no licensed human vaccines or antivirals for treatment of or prevention from infection with encephalitic alphaviruses. Because epidemics are sporadic and unpredictable, and endemic disease is common but rarely diagnosed, it is difficult to identify all populations requiring vaccination; thus, an effective post-exposure treatment method is needed to interrupt ongoing outbreaks. To address this public health need, we have continued development of ML336 to deliver a molecule with prophylactic and therapeutic potential that could be relevant for use in natural epidemics or deliberate release scenario for Venezuelan equine encephalitis virus (VEEV). We report findings from in vitro assessments of four analogs of ML336, and in vivo screening of three of these new derivatives, BDGR-4, BDGR-69 and BDGR-70. The optimal dosing for maximal protection was observed at 12.5 mg/kg/day, twice daily for 8 days. BDGR-4 was tested further for prophylactic and therapeutic efficacy in mice challenged with VEEV Trinidad Donkey (TrD). Mice challenged with VEEV TrD showed 100% and 90% protection from lethal disease when treated at 24 and 48 h post-infection, respectively. We also measured 90% protection for BDGR-4 in mice challenged with Eastern equine encephalitis virus. In additional assessments of BDGR-4 in mice alone, we observed no appreciable toxicity as evaluated by clinical chemistry indicators up to a dose of 25 mg/kg/day over 4 days. In these same mice, we observed no induction of interferon. Lastly, the resistance of VEEV to BDGR-4 was evaluated by next-generation sequencing which revealed specific mutations in nsP4, the viral polymerase.

Sigmodontine community and species responses to El Niño and precipitation in different levels of forest degradation.

Few studies have focused on rodent communities at the margins of an ecoregion or the limits of species' distributions, where the community may be more sensitive to extrinsic variables, both biotic and abiotic. This study evaluates sigmodontine rodent species diversity and overall abundance, and variation associated with climatic variables, in three locations with differing levels of habitat degradation. The study was conducted in northeastern Paraguay, near the western limit of the Upper Paraná Atlantic Forest and near the distributional limits of the three most abundant species in the study sites. Three mark-recapture grids were established and classified as least, moderately and most-degraded based on an analysis of several vegetation parameters. The grids were sampled for five consecutive nights, six times during two years. Shannon diversity and overall abundance were calculated for each sample. Monthly Multivariate ENSO Index and rainfall values were obtained from publicly available resources. Product-moment correlations were calculated between community and climatic parameters, including cumulative values for the climatic variables. The same correlations were calculated for the three common sigmodontine species. 1,632 captures were recorded, representing 13 sigmodontine species. Species richness in the samples (one session on one grid) varied from four to seven. Akodon montensis, Hylaeamys megacephalus and Oligoryzomys nigripes were the three most abundant species. In general, species diversity was negatively correlated with ENSO index, precipitation and precipitation anomaly, including cumulative one- to six-month cumulative values of each. Total sigmodontine abundance was positively correlated with the climatic variables. However, these correlations were not uniform among the three levels of habitat degradation, nor did the three abundant species show similar correlation patterns. The three most abundant species are each near their distributional limits, whereas several less abundant species have distributions that extend well beyond the study area. This somewhat counterintuitive result bears further investigation in other sites at ecoregional margins, to determine whether it is a commonly observed pattern, or an exception. Overall sigmodontine abundances were generally reflective of Akodon montensis abundance, which generally correlated with precipitation (including cumulative amounts). Our analyses of these longitudinal data showed two major effects on sigmodontine species diversity and population. First, they are impacted by habitat and secondly, they are affected by climate (ENSO, precipitation). However, individual species are not impacted similarly. Akodon montensis abundances primarily were correlated with abiotic (climatic) variables, and the correlations were consistent across habitats (biotic factors). In contrast, Hylaeamys megacephalus abundance was correlated with climatic variables in two habitats, but not the moderately-degraded habitat, and Oligoryzomys nigripes abundance was not correlated with climate in the most-degraded habitat. Pocos estudios se han centrado en las comunidades de roedores en los márgenes de una ecorregión o en los límites de las distribuciones de las especies, donde la comunidad puede ser más sensible a las variables extrínsecas, tanto bióticas como abióticas. Este estudio evalúa la diversidad de especies y la abundancia general de roedores sigmodontinos, y la variación asociada con las variables climáticas, en tres lugares con diferentes niveles de degradación del hábitat. Se establecieron tres parcelas de captura-marca-recaptura y se clasificaron como la menos, moderada y más degradadas basadas en un análisis de vegetación de varios parámetros. Las parcelas fueron muestreadas durante cinco noches consecutivas, seis veces durante dos años. La diversidad de Shannon y la abundancia general se calcularon para cada muestra. El índice mensual de ENOS multivariable y los valores de precipitación se obtuvieron de sitios accesibles en Internet. Las correlaciones producto-momento se calcularon entre los parámetros climáticos y de la comunidad, incluidos los valores acumulados para las variables climáticas. Se calcularon las mismas correlaciones para las tres especies comunes de sigmodontinos. Se registraron 1,632 capturas, representando 13 especies sigmodontinos. La riqueza de especies en las muestras (una sesión en una parcela) varió de cuatro a siete. Akodon montensis, Hylaeamys megacephalus y Oligoryzomys nigripes fueron las tres especies más abundantes. En general, la diversidad de especies se correlacionó negativamente con el índice ENOS, la precipitación y la anomalía de la precipitación, incluidos los valores acumulativos de uno a seis meses de cada uno. La abundancia total de sigmodontinos se correlacionó positivamente con las variables climáticas. Sin embargo, estas correlaciones no fueron uniformes entre los tres niveles de degradación del hábitat, ni tampoco entre las tres especies abundantes. Las tres especies más abundantes están cada una cerca de sus límites de distribución, mientras que varias especies menos abundantes tienen distribuciones que se extienden mucho más allá de este sitio. Este resultado algo contraintuitivo conlleva una mayor investigación en otros sitios en los márgenes ecorregionales, para determinar si es un patrón observado comúnmente, o una excepción. Las abundancias de sigmodontinos generalmente reflejaron la abundancia de Akodon montensis, que generalmente se correlacionó con la precipitación (incluidas las cantidades acumuladas). Las conclusiones destacadas de este estudio fueron: (1) diferentes niveles de degradación del hábitat se correlacionan con la variación en la diversidad de especies y la abundancia general de los sigmodontinos, y las especies individuales no muestran los mismos niveles de correlación entre los diferentes hábitats; y (2) la variabilidad climática (ENOS y precipitación) también afecta la diversidad de especies sigmodontinos y la abundancia de la población, y las especies comunes no muestran correlaciones similares entre sí. Las abundancias de Akodon montensis se correlacionaron principalmente con variables abióticas (climáticas), y las correlaciones fueron consistentes en todos los hábitats (factores bióticos). En contraste, la abundancia de Hylaeamys megacephalus se correlacionó con las variables climáticas en dos hábitats, pero no en el hábitat moderadamente degradado, y Oligoryzomys nigripes no se correlacionó con el clima en el hábitat más degradado.

Habitat, species richness and hantaviruses of sigmodontine rodents within the Interior Atlantic Forest, Paraguay.

Four of the nine sigmodontine tribes have species that serve as reservoirs of rodent-borne hantaviruses (RBO-HV), few have been studied in any depth. Several viruses have been associated with human cases of hantavirus pulmonary syndrome often through peridomestic exposure. Jabora (JABV) and Juquitiba (JUQV), harbored by Akodon montensis and Oligoryzomys nigripes, respectively, are endemic and sympatric in the Reserva Natural de Bosque Mbaracayú (RNBM), Paraguay, a protected area of the Interior Atlantic Forest. Rodent communities were surveyed along a 30 km stretch of the RNBM in eight vegetation classifications (Low, High, Bamboo, Riparian and Liana Forests, Bamboo Understory, Cerrado, and Meadow/Grasslands). We collected 417 rodents from which 11 species were identified; Akodon montensis was the predominant species (72%; 95%CI: 64.7%-76.3%), followed by Hylaeamys megacephalus (15% (11.2%-18.2%)) and Oligoryzomys nigripes (9% (6.6%-12.4%)). We examined the statistical associations among habitat (vegetation class) type, rodent species diversity, population structure (age, sex, and weight), and prevalence of RBO-HV antibody and/or viral RNA (Ab/RNA) or characteristic Leishmania tail lesions. Ab/RNA positive rodents were not observed in Cerrado and Low Forest. A. montensis had an overall Ab/RNA prevalence of 7.7% (4.9%-11.3%) and O. nigripes had an overall prevalence of 8.6% (1.8%-23.1%). For A. montensis, the odds of being Ab/RNA positive in High Forest was 3.73 times of the other habitats combined. There was no significant difference among age classes in the proportion of Ab/RNA positive rodents overall (p = 0.66), however, all 11 RNA-positive individuals were adult. Sex and habitat had independent prognostic value for hantaviral Ab/RNA in the study population; age, presence of tail scar/lesion (19% of the rodents) and weight did not. Adjusting for habitat, female rodents had less risk of becoming infected. Importantly, these data suggest habitat preferences of two sympatric rodent reservoirs for two endemic hantaviruses and the importance of including habitat in models of species diversity and habitat fragmentation.

Natural infection of Neotropical bats with hantavirus in Brazil.

Bats (Order: Chiroptera) harbor a high diversity of emerging pathogens presumably because their ability to fly and social behavior favor the maintenance, evolution, and dissemination of these pathogens. Until 2012, there was only one report of the presence of Hantavirus in bats. Historically, it was thought that these viruses were harbored primarily by rodent and insectivore small mammals. Recently, new species of hantaviruses have been identified in bats from Africa and Asia continents expanding the potential reservoirs and range of these viruses. To assess the potential of Neotropical bats as hosts for hantaviruses and its transmission dynamics in nature, we tested 53 bats for active hantaviral infection from specimens collected in Southeastern Brazil. Part of the hantaviral S segment was amplified from the frugivorous Carollia perspicillata and the common vampire bat Desmodus rotundus. DNA sequencing showed high similarity with the genome of Araraquara orthohantavirus (ARQV), which belongs to one of the more lethal hantavirus clades (Andes orthohantavirus). ARQV-like infection was detected in the blood, urine, and organs of D. rotundus. Therefore, we describe a systemic infection in Neotropical bats by a human pathogenic Hantavirus. We also propose here a schematic transmission dynamics of hantavirus in the study region. Our results give insights to new, under-appreciated questions that need to be addressed in future studies to clarify hantavirus transmission in nature and avoid hantavirus outbreaks.

COMPOSITION AND CHARACTERISTICS OF A DIVERSE DIDELPHID COMMUNITY (MAMMALIA: DIDELPHIMORPHIA) IN SUB-TROPICAL SOUTH AMERICA.

Few studies have reported didelphid communities of ≥10 species, and all of these have been from within the tropics sensu stricto of South America. Herein a community of 12 species of didelphids is described from a sub-tropical site in south-central South America. The Reserva Natural del Bosque Mbaracayú, in northeastern Paraguay, lies at the western margin of the Interior Atlantic Forest and the southwestern limit of the Cerrado, two important South American ecoregions. The rich didelphid community in this area likely results from the mosaic of habitats encountered at the distributional limits of these two ecoregions. Within the context of this mosaic, the species' habitat associations and vertical occupancy are discussed, as well as the reproductive patterns and population abundance variation of the more commonly encountered species. Three Monodelphis species were found in sympatry, all strictly terrestrial, along with Cryptonanus chacoensis. Marmosa paraguayana shared all habitats with Gracilinanus agilis, and both of these species primarily were arboreal. Although this natural reserve has been more extensively sampled than any other area in Paraguay, numerous questions remain unanswered regarding this rich didelphid community.

Pocos estudios han registrado comunidades de didélfidos de ≥10 especies, y todas ellas han sido de los trópicos sensu stricto de América del Sur. Aquí reportamos una comunidad de 12 especies de didélfidos de un sitio subtropical en el centro sur de América del Sur. La Reserva Natural del Bosque Mbaracayú, en el noreste de Paraguay, se encuentra en el margen occidental del Bosque Atlántico Interior, y el límite suroeste del Cerrado, dos ecorregiones sudamericanas importantes. La rica comunidad de didélfidos en esta área es probablemente el resultado del mosaico de hábitats encontrados en los límites de distribución de estas dos ecorregiones. Dentro del contexto de este mosaico, discutimos las asociaciones de hábitats y la ocupación vertical de las especies, los patrones reproductivos y la variación de la abundancia de las poblaciones de las especies más comúnmente encontradas. Encontramos tres especies de Monodelphis en simpatría, y todas estrictamente terrestres, junto con Cryptonanus chacoensis. Marmosa paraguayana compartió todos sus hábitats con Gracilinanus agilis, y ambas especies eran principalmente arbóreas. Enfatizamos que a pesar de que esta reserva natural ha sido muestreada más extensamente que cualquier otra área en Paraguay, muchas preguntas siguen sin respuesta con respecto a esta rica comunidad de didélfidos.

Serologic Evidence of Mammarenaviruses among Wild Rodents in Brazil.

We screened blood samples from 560 wild rodents collected in southeastern Brazil for antibodies to a recombinant nucleoprotein (rN) of Junín virus. Six rodents were antibody positive (1.1%), demonstrating evidence of infection with mammarenaviruses in several species of Brazilian rodents.

Evidence of Hantavirus Infection Among Bats in Brazil.

Hantaviruses are zoonotic viruses harbored by rodents, bats, and shrews. At present, only rodent-borne hantaviruses are associated with severe illness in humans. New species of hantaviruses have been recently identified in bats and shrews greatly expanding the potential reservoirs and ranges of these viruses. Brazil has one of the highest incidences of hantavirus cardiopulmonary syndrome in South America, hence it is critical to know what is the prevalence of hantaviruses in Brazil. Although much is known about rodent reservoirs, little is known regarding bats. We captured 270 bats from February 2012 to April 2014. Serum was screened for the presence of antibodies against a recombinant nucleoprotein (rN) of Araraquara virus (ARAQV). The prevalence of antibody to hantavirus was 9/53 with an overall seroprevalence of 17%. Previous studies have shown only insectivorous bats to harbor hantavirus; however, in our study, of the nine seropositive bats, five were frugivorous, one was carnivorous, and three were sanguivorous phyllostomid bats.

Development of (E)-2-((1,4-dimethylpiperazin-2-ylidene)amino)-5-nitro-N-phenylbenzamide, ML336: Novel 2-amidinophenylbenzamides as potent inhibitors of venezuelan equine encephalitis virus.

Venezuelan equine encephalitis virus (VEEV) is an emerging pathogenic alphavirus that can cause significant disease in humans. Given the absence of therapeutic options available and the significance of VEEV as a weaponized agent, an optimization effort was initiated around a quinazolinone screening hit 1 with promising cellular antiviral activity (EC50 = 0.8 µM), limited cytotoxic liability (CC50 > 50 µM), and modest in vitro efficacy in reducing viral progeny (63-fold at 5 µM). Scaffold optimization revealed a novel rearrangement affording amidines, specifically compound 45, which was found to potently inhibit several VEEV strains in the low nanomolar range without cytotoxicity (EC50 = 0.02-0.04 µM, CC50 > 50 µM) while limiting in vitro viral replication (EC90 = 0.17 µM). Brain exposure was observed in mice with 45. Significant protection was observed in VEEV-infected mice at 5 mg kg(-1) day(-1) and viral replication appeared to be inhibited through interference of viral nonstructural proteins.

Discovery of a novel compound with anti-venezuelan equine encephalitis virus activity that targets the nonstructural protein 2.

Alphaviruses present serious health threats as emerging and re-emerging viruses. Venezuelan equine encephalitis virus (VEEV), a New World alphavirus, can cause encephalitis in humans and horses, but there are no therapeutics for treatment. To date, compounds reported as anti-VEEV or anti-alphavirus inhibitors have shown moderate activity. To discover new classes of anti-VEEV inhibitors with novel viral targets, we used a high-throughput screen based on the measurement of cell protection from live VEEV TC-83-induced cytopathic effect to screen a 340,000 compound library. Of those, we identified five novel anti-VEEV compounds and chose a quinazolinone compound, CID15997213 (IC50 = 0.84 µM), for further characterization. The antiviral effect of CID15997213 was alphavirus-specific, inhibiting VEEV and Western equine encephalitis virus, but not Eastern equine encephalitis virus. In vitro assays confirmed inhibition of viral RNA, protein, and progeny synthesis. No antiviral activity was detected against a select group of RNA viruses. We found mutations conferring the resistance to the compound in the N-terminal domain of nsP2 and confirmed the target residues using a reverse genetic approach. Time of addition studies showed that the compound inhibits the middle stage of replication when viral genome replication is most active. In mice, the compound showed complete protection from lethal VEEV disease at 50 mg/kg/day. Collectively, these results reveal a potent anti-VEEV compound that uniquely targets the viral nsP2 N-terminal domain. While the function of nsP2 has yet to be characterized, our studies suggest that the protein might play a critical role in viral replication, and further, may represent an innovative opportunity to develop therapeutic interventions for alphavirus infection.

Phylogenetic exploration of hantaviruses in Paraguay reveals reassortment and host switching in South America.

BACKGROUND: Longitudinal mark-recapture studies of rodents in two sites in the Mbaracayú Biosphere Reserve in the Interior Atlantic Forest of eastern Paraguay have revealed a complex and intriguing pattern of hantaviruses harbored by rodents in this area. Full-length sequencing and phylogenetic analyses were conducted for several rodents from Akodon montensis and Oligoryzomys fornesi. The phylogenetic relationships of these viruses were analyzed in the context of hantaviruses in South America with published S- and M-segment sequences. FINDINGS: Phylogenetic analyses of hantaviruses identified in the Mbaracayú Biosphere Reserve in Paraguay revealed Jabora and Juquitiba viruses are harbored by Akodon montensis and Oligoryzomys fornesi, respectively. These analyses revealed that in general the constituents of the major subclade for the S- and M-segments differ for the South American hantaviruses. Further, the two major groups within subclade C for the M-segment reflect in general the lethality associated with the viruses within each group. CONCLUSIONS: Phylogenetic studies of Jabora and Juquitiba viruses and other Paraguayan viruses in the context of American hantaviruses revealed reassortment and host-switching in the evolution of South American hantaviruses.

Sympatry of 2 hantavirus strains, paraguay, 2003-2007.

To explore geographic and host-taxonomic patterns of hantaviruses in Paraguay, we established sampling sites in the Mbaracayu Biosphere Reserve. We detected Jabora virus and Itapua37/Juquitiba-related virus in locations approximately 20 m apart in different years, which suggested sympatry of 2 distinct hantaviruses.

A habitat-based model for the spread of hantavirus between reservoir and spillover species.

New habitat-based models for spread of hantavirus are developed which account for interspecies interaction. Existing habitat-based models do not consider interspecies pathogen transmission, a primary route for emergence of new infectious diseases and reservoirs in wildlife and man. The modeling of interspecies transmission has the potential to provide more accurate predictions of disease persistence and emergence dynamics. The new models are motivated by our recent work on hantavirus in rodent communities in Paraguay. Our Paraguayan data illustrate the spatial and temporal overlaps among rodent species, one of which is the reservoir species for Jabora virus and others which are spillover species. Disease transmission occurs when their habitats overlap. Two mathematical models, a system of ordinary differential equations (ODE) and a continuous-time Markov chain (CTMC) model, are developed for spread of hantavirus between a reservoir and a spillover species. Analysis of a special case of the ODE model provides an explicit expression for the basic reproduction number, R(0), such that if R(0)<1, then the pathogen does not persist in either population but if R(0)>1, pathogen outbreaks or persistence may occur. Numerical simulations of the CTMC model display sporadic disease incidence, a new behavior of our habitat-based model, not present in other models, but which is a prominent feature of the seroprevalence data from Paraguay. Environmental changes that result in greater habitat overlap result in more encounters among various species that may lead to pathogen outbreaks and pathogen establishment in a new host.