Evolutionary Dynamics of Oropouche Virus in South America.

The Amazon basin is home to numerous arthropod-borne viral pathogens that cause febrile disease in humans. Among these, Oropouche orthobunyavirus (OROV) is a relatively understudied member of the genus Orthobunyavirus, family Peribunyaviridae, that causes periodic outbreaks in human populations in Brazil and other South American countries. Although several studies have described the genetic diversity of the virus, the evolutionary processes that shape the OROV genome remain poorly understood. Here, we present a comprehensive study of the genomic dynamics of OROV that encompasses phylogenetic analysis, evolutionary rate estimates, inference of natural selective pressures, recombination and reassortment, and structural analysis of OROV variants. Our study includes all available published sequences, as well as a set of new OROV genome sequences obtained from patients in Ecuador, representing the first set of genomes from this country. Our results show differing evolutionary processes on the three segments that comprise the viral genome. We infer differing times of the most recent common ancestors of the genome segments and propose that this can be explained by cryptic reassortment. We also present the discovery of previously unobserved putative N-linked glycosylation sites, as well as codons that evolve under positive selection on the viral surface proteins, and discuss the potential role of these features in the evolution of OROV through a combined phylogenetic and structural approach.IMPORTANCE The emergence and reemergence of pathogens such as Zika virus, chikungunya virus, and yellow fever virus have drawn attention toward other cocirculating arboviruses in South America. Oropouche virus (OROV) is a poorly studied pathogen responsible for over a dozen outbreaks since the early 1960s and represents a public health burden to countries such as Brazil, Panama, and Peru. OROV is likely underreported since its symptomatology can be easily confounded with other febrile illnesses (e.g., dengue fever and leptospirosis) and point-of-care testing for the virus is still uncommon. With limited data, there is a need to optimize the information currently available. Analysis of OROV genomes can help us understand how the virus circulates in nature and can reveal the evolutionary forces that shape the genetic diversity of the virus, which has implications for molecular diagnostics and the design of potential vaccines.

ICTV Virus Taxonomy Profile: Peribunyaviridae.

Peribunyaviruses are enveloped and possess three distinct, single-stranded, negative-sense RNA segments comprising 11.2-12.5 kb in total. The family includes globally distributed viruses in the genera Orthobunyavirus, Herbevirus, Pacuvirus and Shangavirus. Most viruses are maintained in geographically-restricted vertebrate-arthropod transmission cycles that can include transovarial transmission from arthropod dam to offspring. Others are arthropod-specific. Arthropods can be persistently infected. Human infection occurs through blood feeding by an infected vector arthropod. Infections can result in a diversity of human and veterinary clinical outcomes in a strain-specific manner. Segment reassortment is evident between some peribunyaviruses. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the taxonomy of the family Peribunyaviridae, which is available at ictv.global/report/peribunyaviridae.

Establishment of a minigenome system for Oropouche virus reveals the S genome segment to be significantly longer than reported previously.

Oropouche virus (OROV) is a medically important orthobunyavirus, which causes frequent outbreaks of a febrile illness in the northern parts of Brazil. However, despite being the cause of an estimated half a million human infections since its first isolation in Trinidad in 1955, details of the molecular biology of this tripartite, negative-sense RNA virus remain limited. We have determined the complete nucleotide sequence of the Brazilian prototype strain of OROV, BeAn 19991, and found a number of differences compared with sequences in the database. Most notable were that the S segment contained an additional 204 nt at the 3' end and that there was a critical nucleotide mismatch at position 9 within the base-paired terminal panhandle structure of each genome segment. In addition, we obtained the complete sequence of the Trinidadian prototype strain TRVL-9760 that showed similar characteristics to the BeAn 19991 strain. By using a T7 RNA polymerase-driven minigenome system, we demonstrated that cDNA clones of the BeAn 19991 L and S segments expressed functional proteins, and also that the newly determined terminal untranslated sequences acted as functional promoters in the minigenome assay. By co-transfecting a cDNA to the viral glycoproteins, virus-like particles were generated that packaged a minigenome and were capable of infecting naive cells.

Genetic analysis of members of the species Oropouche virus and identification of a novel M segment sequence.

Oropouche virus (OROV) is a public health threat in South America, and in particular in northern Brazil, causing frequent outbreaks of febrile illness. Using a combination of deep sequencing and Sanger sequencing approaches, we determined the complete genome sequences of eight clinical isolates that were obtained from patient sera during an Oropouche fever outbreak in Amapa state, northern Brazil, in 2009. We also report the complete genome sequences of two OROV reassortants isolatd from two marmosets in Minas Gerais state, south-east Brazil, in 2012 that contained a novel M genome segment. Interestingly, all 10 isolates possessed a 947 nt S segment that lacked 11 residues in the S-segment 3' UTR compared with the recently redetermined Brazilian prototype OROV strain BeAn19991. OROV maybe circulating more widely in Brazil and in the non-human primate population than previously appreciated, and the identification of yet another reassortant highlights the importance of bunyavirus surveillance in South America.

Genomic and phylogenetic characterization of viruses included in the Manzanilla and Oropouche species complexes of the genus Orthobunyavirus, family Bunyaviridae.

A thorough characterization of the genetic diversity of viruses present in vector and vertebrate host populations is essential for the early detection of and response to emerging pathogenic viruses, yet genetic characterization of many important viral groups remains incomplete. The Simbu serogroup of the genus Orthobunyavirus, family Bunyaviridae, is an example. The Simbu serogroup currently consists of a highly diverse group of related arboviruses that infect both humans and economically important livestock species. Here, we report complete genome sequences for 11 viruses within this group, with a focus on the large and poorly characterized Manzanilla and Oropouche species complexes. Phylogenetic and pairwise divergence analyses indicated the presence of high levels of genetic diversity within these two species complexes, on a par with that seen among the five other species complexes in the Simbu serogroup. Based on previously reported divergence thresholds between species, the data suggested that these two complexes should actually be divided into at least five species. Together these five species formed a distinct phylogenetic clade apart from the rest of the Simbu serogroup. Pairwise sequence divergences among viruses of this clade and viruses in other Simbu serogroup species complexes were similar to levels of divergence among the other orthobunyavirus serogroups. The genetic data also suggested relatively high levels of natural reassortment, with three potential reassortment events present, including two well-supported events involving viruses known to infect humans.

Genetic and biological characterization of selected Changuinola viruses (Reoviridae, Orbivirus) from Brazil.

The genus Orbivirus of the family Reoviridae comprises 22 virus species including the Changuinola virus (CGLV) serogroup. The complete genome sequences of 13 CGLV serotypes isolated between 1961 and 1988 from distinct geographical areas of the Brazilian Amazon region were obtained. All viral sequences were obtained from single-passaged CGLV strains grown in Vero cells. CGLVs are the only orbiviruses known to be transmitted by phlebotomine sandflies. Ultrastructure and molecular analysis by electron microscopy and gel electrophoresis, respectively, revealed viral particles with typical orbivirus size and morphology, as well as the presence of a segmented genome with 10 segments. Full-length nucleotide sequencing of each of the ten RNA segments of the 13 CGLV serotypes provided basic information regarding the genome organization, encoded proteins and genetic traits. Segment 2 (encoding VP2) of the CGLV is uncommonly larger in comparison to those found in other orbiviruses and shows varying sizes even among different CGLV serotypes. Phylogenetic analysis support previous serological findings, which indicate that CGLV constitutes a separate serogroup within the genus Orbivirus. In addition, six out of 13 analysed CGLV serotypes showed reassortment of their genome segments.

Diversity and distribution of hantaviruses in South America.

Hantaviruses are important contributors to disease burden in the New World, yet many aspects of their distribution and dynamics remain uncharacterized. To examine the patterns and processes that influence the diversity and geographic distribution of hantaviruses in South America, we performed genetic and phylogeographic analyses of all available South American hantavirus sequences. We sequenced multiple novel and previously described viruses (Anajatuba, Laguna Negra-like, two genotypes of Castelo dos Sonhos, and two genotypes of Rio Mamore) from Brazilian Oligoryzomys rodents and hantavirus pulmonary syndrome cases and identified a previously uncharacterized species of Oligoryzomys associated with a new genotype of Rio Mamore virus. Our analysis indicates that the majority of South American hantaviruses fall into three phylogenetic clades, corresponding to Andes and Andes-like viruses, Laguna Negra and Laguna Negra-like viruses, and Rio Mamore and Rio Mamore-like viruses. In addition, the dynamics and distribution of these viruses appear to be shaped by both the geographic proximity and phylogenetic relatedness of their rodent hosts. The current system of nomenclature used in the hantavirus community is a significant impediment to understanding the ecology and evolutionary history of hantaviruses; here, we suggest strict adherence to a modified taxonomic system, with species and strain designations resembling the numerical system of the enterovirus genus.

Genomic and phylogenetic characterization of Brazilian yellow fever virus strains.

Globally, yellow fever virus infects nearly 200,000 people, leading to 30,000 deaths annually. Although the virus is endemic to Latin America, only a single genome from this region has been sequenced. Here, we report 12 Brazilian yellow fever virus complete genomes, their genetic traits, phylogenetic characterization, and phylogeographic dynamics. Variable 3' noncoding region (3'NCR) patterns and specific mutations throughout the open reading frame altered predicted secondary structures. Our findings suggest that whereas the introduction of yellow fever virus in Brazil led to genotype I-predominant dispersal throughout South and Central Americas, genotype II remained confined to Bolivia, Peru, and the western Brazilian Amazon.

Circular agriculture increases food production and can reduce N fertilizer use of commercial farms for tropical environments.

World food production must increase in the coming years with minimal environmental impact for food and nutrition security. Circular Agriculture has emerged as an approach to minimize non-renewable resource depletion and encourage by-product reuse. The goal of this study was to evaluate Circular Agriculture as a tool to increase food production and N recovery. The assessment was conducted for two Brazilian farms (Farm 1; Farm 2) with Oxisols under no-till and a diversified cropping system, including five species of grain, three cover crop species, and sweet potato. Both farms implemented an annual two-crop rotation and an integrated crop-livestock system with beef cattle confined for 2-years. Grain and forage from the fields, leftovers from silos, and crop residues were used as cattle feed. Grain yield was 4.8 and 4.5 t ha-1 for soybean, 12.5 and 12.1 t ha-1 for maize, and 2.6 and 2.4 t ha-1 for common bean, for Farm 1 and Farm 2, respectively, which is higher than the national average. The animals gained 1.2 kg day-1 of live weight. Farm 1 exported 246 kg ha-1 year-1 of N in grains, tubers, and animals, while 216 kg ha-1 year-1 was added as fertilizer and N to cattle. Farm 2 exported 224 kg ha-1 year-1 in grain and animals, while 215 kg ha-1 year-1 was added as fertilizer and N to cattle. Circular practices, i.e., no-till, crop rotation, year-round soil covered, maize intercropped with brachiaria ruziziensis, biological N fixation, and crop-livestock integration, increased crop yield and decreased N application by 14.7 % (Farm 1) and 4.3 % (Farm 2). 85 % of the N consumed by the confined animals was excreted and converted into organic compost. Overall, circular practices associated with adequate crop management allowed recovering high rate of applied N, reducing environmental impacts, and increasing food production with lower production costs.

Molecular epidemiology of Laguna Negra virus, Mato Grosso State, Brazil.

We associated Laguna Negra virus with hantavirus pulmonary syndrome in Mato Grosso State, Brazil, and a previously unidentified potential host, the Calomys callidus rodent. Genetic testing revealed homologous sequencing in specimens from 20 humans and 8 mice. Further epidemiologic studies may lead to control of HPS in Mato Grosso State.

Genetic characterization of the Wyeomyia group of orthobunyaviruses and their phylogenetic relationships.

Phylogenetic analyses can give new insights into the evolutionary history of viruses, especially of viruses with segmented genomes. However, sequence information for many viral families or genera is still limited and phylogenies based on single or short genome fragments can be misleading. We report the first genetic analysis of all three genome segments of Wyeomyia group viruses Wyeomyia, Taiassui, Macaua, Sororoca, Anhembi and Cachoeira Porteira (BeAr328208) in the genus Orthobunyavirus of the family Bunyaviridae. In addition, Tucunduba and Iaco viruses were identified as members of the Wyeomyia group. Features of Wyeomyia group members that distinguish them from other viruses in the Bunyamwera serogroup and from other orthobunyaviruses, including truncated NSs sequences that may not counteract the host's interferon response, were characterized. Our findings also suggest genome reassortment within the Wyeomyia group, identifying Macaua and Tucunduba viruses as M-segment reassortants that, in the case of Tucunduba virus, may have altered pathogenicity, stressing the need for whole-genome sequence information to facilitate characterization of orthobunyaviruses and their phylogenetic relationships.

Characterization of the Candiru antigenic complex (Bunyaviridae: Phlebovirus), a highly diverse and reassorting group of viruses affecting humans in tropical America.

The genus Phlebovirus of the family Bunyaviridae consists of approximately 70 named viruses, currently assigned to nine serocomplexes (species) based on antigenic similarities. Sixteen other named viruses that show little serologic relationship to the nine recognized groups are also classified as tentative species in the genus. In an effort to develop a more precise classification system for phleboviruses, we are attempting to sequence most of the named viruses in the genus with the goal of clarifying their phylogenetic relationships. In this report, we describe the serologic and phylogenetic relationships of 13 viruses that were found to be members of the Candiru serocomplex; 6 of them cause disease in humans. Analysis of full genome sequences revealed branching inconsistencies that suggest five reassortment events, all involving the M segment, and thus appear to be natural reassortants. This high rate of reassortment illustrates the inaccuracy of a classification system based solely on antigenic relationships.

Nitrate losses and nitrous oxide emissions under contrasting tillage and cover crop management.

Agroecosystems in the upper Mississippi River Basin are highly productive but often contribute to deterioration of water quality and greenhouse gas emissions. Cover cropping and no-till are conservation strategies implemented to reduce the environmental impact of these agroecosystems. However, using multiple strategies can lead to systemwide interactions that are not fully understood. These interactions can affect not only environmental quality metrics, such as subsurface drainage nitrate losses or nitrous oxide (N2 O) emissions, but also may influence crop production potential. A field trial was initiated comparing nitrate losses, N2 O emissions, and crop production under systems with fall chisel plow tillage, fall chisel plow tillage with an oat (Avena sativa L.) cover crop (CP-oat), no-till (NT), no-till with a rye (Secale cereale L.) cover crop (NT-rye), and NT with zero N fertilizer. Pathways for nitrate losses and N2 O emissions did not appear linked and were not tied to cover crop or tillage practices. Nitrate losses were linked with drainage volumes, and cover crops and tillage had limited effect on cumulative drainage volumes. Notably, NT-rye altered the relationship between drainage volume and nitrate losses by reducing nitrate concentrations, lowering nitrate losses by 59 ±9% compared with CP-oat and 67 ± 9% compared with NT. Neither cover crop nor tillage consistently affected N2 O emissions or crop yield. Rather, N2 O emissions were closely tied with fertilizer N application and seasonal weather patterns. These findings indicate that nitrate leaching and N2 O emissions are regulated by separate mechanisms, so conservation management may require stacking multiple practices to be effective.

Both subsurface nitrate losses and nitrous oxide emissions were linked with weather. Subsurface nitrate losses were linked with cumulative annual drainage. Nitrous oxide emissions were linked with fertilizer N applications. Rye cover crop with no-till reduced nitrate losses with no yield declines.

Molecular epidemiology of Oropouche virus, Brazil.

Oropouche virus (OROV) is the causative agent of Oropouche fever, an urban febrile arboviral disease widespread in South America, with >30 epidemics reported in Brazil and other Latin American countries during 1960-2009. To describe the molecular epidemiology of OROV, we analyzed the entire N gene sequences (small RNA) of 66 strains and 35 partial Gn (medium RNA) and large RNA gene sequences. Distinct patterns of OROV strain clustered according to N, Gn, and large gene sequences, which suggests that each RNA segment had a different evolutionary history and that the classification in genotypes must consider the genetic information for all genetic segments. Finally, time-scale analysis based on the N gene showed that OROV emerged in Brazil ≈223 years ago and that genotype I (based on N gene data) was responsible for the emergence of all other genotypes and for virus dispersal.

Pygmy rice rat as potential host of Castelo dos Sonhos Hantavirus.

To study the dynamics of wild rodent populations and identify potential hosts for hantavirus, we conducted an eco-epidemiologic study in Campo Novo do Parecis, Mato Grosso State, Brazil. We detected and genetically characterized Castelo dos Sonhos virus found in a species of pygmy rice rat (Oligoryzomys utiaritensis).

Hantaviruses and hantavirus pulmonary syndrome, Maranhao, Brazil.

To confirm circulation of Anajatuba virus in Maranhao, Brazil, we conducted a serologic survey (immunoglobulin G ELISA) and phylogenetic studies (nucleocapsid gene sequences) of hantaviruses from wild rodents and persons with hantavirus pulmonary syndrome. This virus is transmitted by Oligoryzomys fornesi rodents and is responsible for hantavirus pulmonary syndrome in this region.

Molecular epidemiology of Saint Louis encephalitis virus in the Brazilian Amazon: genetic divergence and dispersal.

Saint Louis encephalitis virus (SLEV), a member of the genus Flavivirus (family Flaviviridae), is an encephalitogenic arbovirus broadly distributed in the Americas. Phylogenetic analysis based on the full-length E gene sequences obtained for 30 Brazilian SLEV strains was performed using different methods including Bayesian and relaxed molecular clock approaches. A new genetic lineage was suggested, hereafter named genotype VIII, which co-circulates with the previously described genotype V in the Brazilian Amazon region. Genotypes II and III were restricted to São Paulo state (South-east Atlantic rainforest ecosystem). The analysis also suggested the emergence of an SLEV common ancestor between 1875 and 1973 (mean of 107 years ago), giving rise to two major genetic groups: genotype II, more prevalent in the North America, and a second group comprising the other genotypes (I and III-VIII), broadly dispersed throughout the Americas, suggesting that SLEV initially emerged in South America and spread to North America. In conclusion, the current study demonstrates the high genetic variability of SLEV and its geographical dispersion in Brazil and other New World countries.

Circulation of hantaviruses in the influence area of the Cuiabá-Santarém Highway.

We describe evidence of circulation of hantaviruses in the influence area of the Santarém-Cuiabá Highway (BR-163) in the Brazilian Amazon through the prevalence of specific antibodies against hantaviruses in inhabitants living in four municipalities of this area: Novo Progresso (2.16%) and Trairão (4.37%), in state of Pará (PA), and Gua-rantã do Norte (4.74%) and Marcelândia (9.43%), in state of Mato Grosso. We also demonstrate the ongoing association between Castelo dos Sonhos virus (CASV) and hantavirus pulmonary syndrome (HPS) cases in the Castelo dos Sonhos district (municipality of Altamira, PA) and the first report of CASV in the municipalities of Novo Progresso and Guarantã do Norte. The results of this work highlight the risk for a possible increase in the number of HPS cases and the emergence of new hantavirus lineages associated with deforestation in this Amazonian area after the conclusion of paving works on BR-163 Highway.