Lessons from a Multilaboratorial Task Force for Diagnosis of a Fatal Toxoplasmosis Outbreak in Captive Primates in Brazil.

Toxoplasmosis is an important zoonotic disease caused by the parasite Toxoplasma gondii and is especially fatal for neotropical primates. In Brazil, the Ministry of Health is responsible for national epizootic surveillance, but some diseases are still neglected. Here, we present an integrated investigation of an outbreak that occurred during the first year of the COVID-19 pandemic among eleven neotropical primates housed at a primatology center in Brazil. After presenting non-specific clinical signs, all animals died within four days. A wide range of pathogens were evaluated, and we successfully identified T. gondii as the causative agent within four days after necropsies. The liver was the most affected organ, presenting hemorrhage and hepatocellular necrosis. Tachyzoites and bradyzoite cysts were observed in histological examinations and immunohistochemistry in different organs; in addition, parasitic DNA was detected through PCR in blood samples from all specimens evaluated. A high prevalence of Escherichia coli was also observed, indicating sepsis. This case highlights some of the obstacles faced by the current Brazilian surveillance system. A diagnosis was obtained through the integrated action of researchers since investigation for toxoplasmosis is currently absent in national guidelines. An interdisciplinary investigation could be a possible model for future epizootic investigations in animals.

Seroprevalence of Hantavirus among Manual Cane Cutters and Epidemiological Aspects of HPS in Central Brazil.

Hantavirus pulmonary syndrome (HPS) is a rodent-borne zoonotic disease that is endemic throughout the Americas. Agricultural activities increase exposure to wild rodents, especially for sugarcane cutters. We carried out a survey of the epidemiological aspects of HPS and investigated the prevalence of hantavirus infection in the sugarcane cutter population from different localities in the Brazilian Midwest region. We conducted a retrospective study of all confirmed HPS cases in the state of Goiás reported to the National HPS surveillance system between 2007 and 2017, along with a seroepidemiological study in a population of sugarcane cutters working in Goiás state in 2016, using the anti-hantavirus (Andes) ELISA IgG. A total of 634 serum samples from cane cutters were tested for hantavirus antibodies, with 44 (6.9%) being IgG-reactive according to ELISA. The destination of garbage was the only statistically significant variable (p = 0.03) related to the detection of hantavirus IgG (p < 0.05). We described the epidemiological profile of reported hantavirus cases in Goiás-a highly endemic area for HPS, and where the seroepidemiological study was conducted. Our results increase our knowledge about hantavirus infections in Brazil and highlight the vulnerability of sugarcane cutters to a highly lethal disease that, to date, has no specific treatment or vaccination.

Case Report: Hantavirus Cardiopulmonary Syndrome Diagnostic in the Face of the COVID-19 Pandemic.

Hantavirus cardiopulmonary syndrome (HCPS) is an emerging rodent-borne disease in the Americas. The most common initial symptoms of HCPS are similar to those of COVID-19 and other respiratory infections that evolve rapidly to respiratory failure, resulting from pulmonary edema and shock in about 40% of cases. We describe a fatal case of HCPS in a 24-year-old man who was hospitalized with fever, hemoconcentration, thrombocytopenia, leukocytosis, dry cough and a bilateral diffuse alveolar pulmonary infiltrate during the emergence of the COVID-19 pandemic in Brazil. HCPS needs to be ruled out in patients with clinical manifestations compatible with respiratory infections such as influenza and COVID-19.

The Serological Cross-Detection of Bat-Borne Hantaviruses: A Valid Strategy or Taking Chances?

Bats are hosts of a range of viruses, and their great diversity and unique characteristics that distinguish them from all other mammals have been related to the maintenance, evolution, and dissemination of these pathogens. Recently, very divergent hantaviruses have been discovered in distinct species of bats worldwide, but their association with human disease remains unclear. Considering the low success rates of detecting hantavirus RNA in bat tissues and that to date no hantaviruses have been isolated from bat samples, immunodiagnostic tools could be very helpful to understand pathogenesis, epidemiology, and geographic range of bat-borne hantaviruses. In this sense, we aimed to identify in silico immunogenic B-cell epitopes present on bat-borne hantaviruses nucleoprotein (NP) and verify if they are conserved among them and other selected members of Mammantavirinae, using a combination of (the three most used) different prediction algorithms, ELLIPRO, Discotope 2.0, and PEPITO server. To support our data, we in silico modeled 3D structures of NPs from representative members of bat-borne hantaviruses, using comparative and ab initio methods due to the absence of crystallographic structures of studied proteins or similar models in the Protein Data Bank. Our analysis demonstrated the antigenic complexity of the bat-borne hantaviruses group, showing a low sequence conservation of epitopes among members of its own group and a minor conservation degree in comparison to Orthohantavirus, with a recognized importance to public health. Our data suggest that the use of recombinant rodent-borne hantavirus NPs to cross-detect antibodies against bat- or shrew-borne viruses could underestimate the real impact of this virus in nature.

Orthohantavirus Survey in Indigenous Lands in a Savannah-Like Biome, Brazil.

In Brazil, the first confirmed cases of hantavirus cardiopulmonary syndrome in Indigenous populations occurred in 2001. The purpose of this study was to determine the seroprevalence of orthohantavirus infections in the Utiariti Indigenous land located in the southeastern region of the Brazilian Amazon. In December 2014 and 2015, a survey was conducted using an enzyme-linked immunosorbent assay in nine villages belonging to the Haliti-Paresí Indigenous communities. A total of 301 participants were enrolled in the study. Of the two study cohorts, the one from 2014 showed a prevalence of 12.4%, whereas the one from 2015 had a serum prevalence of 13.4%. Analysis of the paired samples of 110 Indigenous people who participated in both stages of the study enabled identification of four individuals who had seroconverted during the study period. Identifying the circulation of orthohantaviruses in the Utiariti Indigenous land highlights a serious public health problem in viral expansion and highlights the need to implement preventive measures appropriate to the sociocultural reality of these communities.

Aporé virus, a novel mammarenavirus (Bunyavirales: Arenaviridae) related to highly pathogenic virus from South America.

Here, we report the complete genome sequence of the Aporé virus (Bunyavirales: Arenaviridae), obtained from a wild rodent Oligoryzomys mattogrossae captured in Mato Grosso do Sul state, Brazil. The genome of this virus showed strong similarity to highly pathogenic mammarenavirus from South America.

The mystery of the phylogeographic structural pattern in rodent-borne hantaviruses.

Hantaviruses (order Bunyavirales, family Hantaviridae) are important zoonotic pathogens. Because of the great diversity of their reservoir hosts, hantaviruses are excellent models to evaluate the dynamics of virus-host co-evolution. To understand the mechanisms behind the evolutionary history of hantaviruses through virus-reservoir interactions, it is important to know how the radiation and diversity of hantaviruses occurred. In this paper, we evaluate the pattern of hantavirus diversification based on a complete S segment representing major groups of hantaviruses found in the Americas. Phylogenetic analyses revealed a high degree of phylogeographic structure and a surprising pattern of geographical distribution of New World hantaviruses. The available data suggest that hantaviruses related to the Arvicolinae rodent subfamily in North America probably emerged and initially adapted from a shared common ancestor of the Tula virus. The first clade of hantaviruses associated with Neotominae occupied a stem lineage, especially those that emerged in Central America or Mexico. Hantaviruses from Central America and Mexico found in Neotominae rodents spread northward and probably gave rise to the first phylogroup of hantaviruses associated with Sigmodontinae in North America. Two preferential host-switching transmissions in hantaviruses apparently gave rise to two different paraphyletic group in Neotominae and Sigmodontinae. Our study supports a probable epicenter of diversification in Central America and/or Mexico for hantaviruses related to both the Neotominae and Sigmodontinae subfamilies.

Aporé virus, a novel mammarenavirus (Bunyavirales: Arenaviridae) related to highly pathogenic virus from South America

Here, we report the complete genome sequence of the Aporé virus (Bunyavirales: Arenaviridae), obtained from a wild rodent Oligoryzomys mattogrossae captured in Mato Grosso do Sul state, Brazil. The genome of this virus showed strong similarity to highly pathogenic mammarenavirus from South America.

Expansion of the range of Necromys lasiurus (Lund, 1841) into open areas of the Atlantic Forest biome in Rio de Janeiro state, Brazil, and the role of the species as a host of the hantavirus.

Necromys lasiurus is a generalist rodent that is thought to be the main reservoir of the Araraquara hantavirus, which causes Hantavirus Pulmonary Syndrome, in the Brazilian Cerrado savanna. This species occurs naturally in the open habitats of the Cerrado, Pantanal and Caatinga biomes, where it often occurs at high densities, although the distribution of the species has recently been observed expanding into the Brazilian state of Rio de Janeiro. This study aimed to map the occurrence of N. lasiurus within the Atlantic Forest of Rio de Janeiro state and discuss the potential role of the species as a reservoir of the Araraquara hantavirus in these areas. The study was based on a comprehensive literature search and four expeditions for the collection of specimens in the state of Rio de Janeiro. The data were used to predict the distribution of N. lasiurus, confirm the distribution of the species in the state, and detect the rates of hantavirus infection in these rodents. Necromys lasiurus has been recorded at 16 localities in 10 municipalities of Rio de Janeiro state. The relative abundance of N. lasiurus was low at all localities, except for the REBIO Poço das Antas and APA-BRSJ, two protected areas. Necromys lasiurus was associated primarily with landscapes dominated by farmland (plantations or pasture) at relatively low altitudes in the vicinity of bodies of water. A total of 204 serum samples were collected, but none were reactive for hantavirus. The distribution of N. lasiurus is expanding into many areas of the anthropogenic matrix, but it is not usually either abundant or dominant in these areas. The relatively reduced abundance of N. lasiurus in Rio de Janeiro and the lack of infection in all the areas investigated indicate that it is unlikely to be a reservoir of hantavirus in this region in the near future.

Xapuri virus, a novel mammarenavirus: natural reassortment and increased diversity between New World viruses.

Mammarenavirus RNA was detected in Musser's bristly mouse (Neacomys musseri) from the Amazon region, and this detection indicated that rodents were infected with a novel mammarenavirus, with the proposed name Xapuri virus (XAPV), which is phylogenetically related to New World Clade B and Clade C viruses. XAPV may represent the first natural reassortment of the Arenaviridae family and a new unrecognized clade within the Tacaribe serocomplex group.

Co-circulation of Araraquara and Juquitiba Hantavirus in Brazilian Cerrado.

Hantavirus cardiopulmonary syndrome is an emerging serious disease in the Americas, transmitted from wild rodents to humans through inhalation of aerosol containing virus. Herein, we characterized two distinct hantaviruses circulating in rodent species form Central Plateau, Midwestern region of Brazil in the Cerrado (savanna-like) biome, an area characterized by small trees and grasses adapted to climates with long dry periods. In this study, we identified the co-circulation of the Araraquara virus and a possible new lineage of the Juquitiba virus (JUQV) in Oligoryzomys nigripes. The implications of co-circulation are still unknown, but it can be the key for increasing viral diversity or emergence of new species through spillover or host switching events leading to co-infection and consequently recombination or reassortment between different virus species. Phylogenetic analyses based on the complete S segment indicated that, alongside with Oligoryzomys mattogrossae rodents, O. nigripes species could also have a whole as JUQV reservoir in the Cerrado biome. Although these rodents' species are common in the Cerrado biome, they are not abundant demonstrating how complex and different hantavirus enzootic cycles can be in this particular biome.

Detection of Latino virus (Arenaviridae: Mammarenavirus) naturally infecting Calomys callidus.

Mammarenavirus species are associated with a specific rodent host species, although an increasing number of virus has been associated to more than one host, suggesting that co-evolution is less robust than initially thought. There are few eco-epidemiological studies of South America mammarenaviruses in non-endemic areas of Arenavirus Hemorrhagic Fever, affecting specially our current knowledge about animal reservoirs and virus range and host-virus relations. In Brazil, seven arenavirus species were described in seven different rodent species. Here in we describe a new rodent reservoir species in Brazil related to the previously described Latino mammarenavirus (LATV) MARU strain. Samples of 148 rodents from Mato Grosso state, Brazil were analyzed. Amplification of the glycoprotein precursor gene (GPC) was observed in six Calomys callidus rodents. According to phylogenetic inferences, is observed a well-supported monophyletic clade of LATV from C. callidus and other Clade C mammarenavirus. In addition, the phylogenetic relations of both genes showed a close relation between LATV MARU and Capão Seco strains, two distinct lineages. Additionally, the results obtained in this study point out to a change of scenario and in previously stabilized patterns in the dynamics of South American mammarenaviruses, showing that with more studies in AHF non-endemic or silent areas, more potential hosts for this virus will be discovered.

A Fatal Hantavirus Pulmonary Syndrome Misdiagnosed as Dengue: An Investigation into the First Reported Case in Rio de Janeiro State, Brazil.

We report the results of an investigation into a fatal case of hantavirus pulmonary syndrome (HPS) in Rio de Janeiro State, Brazil, where the disease had not been reported previous to 2015. Following the notification of an HPS case, serum samples were collected from the household members and work contacts of the HPS patient and tested for antibody to hantaviruses. Seroprevalence of 22% (10/45) was indicated for hantavirus out of 45 human samples tested. Blood and tissue samples were collected from 72 rodents during fieldwork to evaluate the prevalence of hantavirus infection, by using enzyme-linked immunosorbent assay IgG, and to characterize the rodent hantavirus reservoir(s), by reverse transcription polymerase chain reaction and sequencing. Antibody prevalence was 6.9%. The circulation of a single genotype, the Juquitiba hantavirus, carried by two rodent species, black-footed pigmy rice rat (Oligoryzomys nigripes) and cursor grass mouse (Akodon cursor), was shown by analysis of the nucleotide sequences of the S segment. Juquitiba hantavirus circulates in rodents of various species, but mainly in the black-footed pigmy rice rat. HPS is a newly recognized clinical entity in Rio de Janeiro State and should be considered in patients with febrile illness and acute respiratory distress.

New bunya-like viruses: Highlighting their relations.

The standard virus classification scheme for arenaviruses and bunyaviruses shifted dramatically when several groups reported the detection and isolation of divergent groups of viruses in a variety of insect collections. Although these viral families can differ in terms of morphology, structure and genetics, recent findings indicate these viruses may have a shared evolutionary origin. To determine the phylogenetic relations among these families, we inferred phylogenetic trees using three methods. The Maximum Likelihood and Bayesian trees were rooted as suggested by the (molecular clock-rooted) BEAST tree. Our results highlight a noteworthy relation among these viral supergroups of different genome organizations. Our study suggests that the best scenario is the existence of at least three monophyletic supergroups, all of them well supported. The recent data indicate that these viruses are evolutionarily and genetically interconnected. While these supergroups appear to be closely related in our phylogenetic analysis, other viruses should be investigated in future research. In sum, our results also provide insights into the classification scheme, thereby providing a new perspective about the fundamental questions of family origins, diversity and genome evolution.

Microorganisms in ticks (Acari: Ixodidae) collected on marsupials and rodents from Santa Catarina, Paraná and Mato Grosso do Sul states, Brazil.

Information about tick fauna and monitoring of pathogen prevalences in ticks (Acari: Ixodidae) in various habitat types can enhance knowledge about the epidemiology of tick-borne pathogens in Brazil. This work shows the results of a study of tick parasitism of wild rodents and marsupials collected in seven localities in the southern part of Brazil, within Atlantic Forest and Cerrado biomes. A total of 61 ticks were collected from small mammals, and after identification to the species level, the ticks were individually tested for the presence of bacteria of the genera Rickettsia, Borrelia, family Anaplasmataceae, and protozoa of the genus Babesia. The following species of ticks were found: Amblyomma ovale Koch, 1844, Amblyomma dubitatum Neumann, 1899, Amblyomma fuscum Neumann, 1907, Ixodes aragaoi Fonseca, 1935, Ixodes fuscipes Koch, 1844, Ixodes loricatus Neumann, 1899, and Ixodes schulzei Aragão and Fonseca, 1951. Among tested ticks, no DNA of Borrelia, Babesia or Anaplasmataceae was detected. Two nymphs of A. ovale were found infected with Rickettsia bellii and four nymphs of I. aragaoi with Rickettsia sp., genetically close to Rickettsia monacensis, Rickettsia tamurae and the endosymbiont Rickettsia spp., previously found in various Ixodidae. In one nymph of A. fuscum, DNA of a novel Hepatozoon sp. was found. Additionally we provide novel tick-host associations.

Comparison of the nutritional content of products, with and without nutrient claims, targeted at children in Brazil.

Many children's food products highlight positive attributes on their front-of-package labels in the form of nutrient claims. This cross-sectional study investigated all retailed packaged foods (n 5620) in a major Brazilian supermarket, in order to identify the availability of products targeted at children, and to compare the nutritional content of products with and without nutrient claims on labels. Data on energy, carbohydrate, protein, fibre, Na and total and SFA content, along with the presence and type of nutrient claims, were obtained in-store from labels of all products. Products targeted at children were identified, divided into eight food groups and compared for their nutritional content per 100 g/ml and the presence of nutrient claims using the Mann-Whitney U test (P<0·05). Of the 535 food products targeted at children (9·5 % of all products), 270 (50·5 %) displayed nutrient claims on their labels. Children's products with nutrient claims had either a similar or worse nutritional content than their counterparts without nutrient claims. The major differences among groups were found in Group 8 (e.g. sauces and ready meals), in which children's products bearing nutrient claims had higher energy, carbohydrate, Na and total and SFA content per 100 g/ml than products without nutrient claims (P<0·05). This suggests that, to prevent misleading parents who are seeking healthier products for their children, the regulation on the use of nutrient claims should be revised, so that only products with appropriate nutrient profiles are allowed to display them.

Zoonotic Bartonella species in wild rodents in the state of Mato Grosso do Sul, Brazil.

Several rodent-associated Bartonella species cause disease in humans but little is known about their epidemiology in Brazil. The presence of Bartonella spp. in wild rodents captured in two municipalities of the Mato Grosso do Sul state was assessed by polymerase chain reaction (PCR). Fragments of heart tissue from 42 wild rodents were tested using primers targeting the Bartonella 16S-23S intergenic transcribed spacer (ITS) region and citrate synthase gltA gene. The wild rodents were identified based on external and cranial morphology and confirmed at species level by mitochondrial DNA (cytochrome B) sequencing and karyotype. Overall, 42.9% (18/42) of the wild rodents were PCR positive for Bartonella spp.: Callomys callosus (04), Cerradomys maracajuensis (04), Hylaeamus megacephalus (01), Necromys lasiurus (06), Nectomys squamipes (01), Oecomys catherinae (01) and Oxymycterus delator (01). Bartonella vinsonii subsp. arupensis was detected in N. lasiurus (46%) and C. callosus (21%) captured in the two study sites. We reported the first molecular detection of B. vinsonii subsp. arupensis in different species of wild rodents collected in the Brazilian territory. Further studies are needed to examine the role of these mammals in the eco-epidemiology of bartonellosis in Brazil.

Detection of different South American hantaviruses.

Hantaviruses are the etiologic agents of Hemorrhagic Fever with Renal Syndrome (HFRS) in Old World, and Hantavirus Pulmonary Syndrome (HPS)/Hantavirus Cardiopulmonary Syndrome (HCPS), in the New World. Serological methods are the most common approach used for laboratory diagnosis of HCPS, however theses methods do not allow the characterization of viral genotypes. The polymerase chain reaction (PCR) has been extensively used for diagnosis of viral infections, including those caused by hantaviruses, enabling detection of few target sequence copies in the sample. However, most studies proposed methods of PCR with species-specific primers. This study developed a simple and reliable diagnostic system by RT-PCR for different hantavirus detection. Using new primers set, we evaluated human and rodent hantavirus positive samples of various regions from Brazil. Besides, we performed computational analyzes to evaluate the detection of other South American hantaviruses. The diagnostic system by PCR proved to be a sensible and simple assay, allowing amplification of Juquitiba virus, Araraquara virus, Laguna Negra virus, Rio Mamore virus and Jabora virus, beyond of the possibility of the detecting Andes, Anajatuba, Bermejo, Choclo, Cano Delgadito, Lechiguanas, Maciel, Oran, Pergamino and Rio Mearim viruses. The primers sets designed in this study can detect hantaviruses from almost all known genetics lineages in Brazil and from others South America countries and also increases the possibility to detect new hantaviruses. These primers could easily be used both in diagnosis of suspected hantavirus infections in humans and also in studies with animals reservoirs.