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Separation of Plasmodium falciparum Late Stage-infected Erythrocytes by Magnetic Means
No full textUnlike other Plasmodium species, P. falciparum can be cultured in the lab, which facilitates its study 1. While the parasitemia achieved can reach the ≈40% limit, the investigator usually keeps the percentage at around 10%. In many cases it is necessary to isolate the parasite-containing red blood cells (RBCs) from the uninfected ones, to enrich the culture and proceed with a given experiment. When P. falciparum infects the erythrocyte, the parasite degrades and feeds from haemoglobin 2, 3. However, the parasite must deal with a very toxic iron-containing haem moiety 4, 5. The parasite eludes its toxicity by transforming the haem into an inert crystal polymer called haemozoin 6, 7. This iron-containing molecule is stored in its food vacuole and the metal in it has an oxidative state which differs from the one in haem 8. The ferric state of iron in the haemozoin confers on it a paramagnetic property absent in uninfected erythrocytes. As the invading parasite reaches maturity, the content of haemozoin also increases 9, which bestows even more paramagnetism on the latest stages of P. falciparum inside the erythrocyte. Based on this paramagnetic property, the latest stages of P. falciparum infected-red blood cells can be separated by passing the culture through a column containing magnetic beads. These beads become magnetic when the columns containing them are placed on a magnet holder. Infected RBCs, due to their paramagnetism, will then be trapped inside the column, while the flow-through will contain, for the most part, uninfected erythrocytes and those containing early stages of the parasite. Here, we describe the methodology to enrich the population of late stage parasites with magnetic columns, which maintains good parasite viability 10. After performing this procedure, the unattached culture can be returned to an incubator to allow the remaining parasites to continue growing.Unlike other Plasmodium species, P. falciparum can be cultured in the lab, which facilitates its study 1. While the parasitemia achieved can reach the ≈40% limit, the investigator usually keeps the percentage at around 10%. In many cases it is necessary to isolate the parasite-containing red blood cells (RBCs) from the uninfected ones, to enrich the culture and proceed with a given experiment. When P. falciparum infects the erythrocyte, the parasite degrades and feeds from haemoglobin 2, 3. However, the parasite must deal with a very toxic iron-containing haem moiety 4, 5. The parasite eludes its toxicity by transforming the haem into an inert crystal polymer called haemozoin 6, 7. This iron-containing molecule is stored in its food vacuole and the metal in it has an oxidative state which differs from the one in haem 8. The ferric state of iron in the haemozoin confers on it a paramagnetic property absent in uninfected erythrocytes. As the invading parasite reaches maturity, the content of haemozoin also increases 9, which bestows even more paramagnetism on the latest stages of P. falciparum inside the erythrocyte. Based on this paramagnetic property, the latest stages of P. falciparum infected-red blood cells can be separated by passing the culture through a column containing magnetic beads. These beads become magnetic when the columns containing them are placed on a magnet holder. Infected RBCs, due to their paramagnetism, will then be trapped inside the column, while the flow-through will contain, for the most part, uninfected erythrocytes and those containing early stages of the parasite. Here, we describe the methodology to enrich the population of late stage parasites with magnetic columns, which maintains good parasite viability 10. After performing this procedure, the unattached culture can be returned to an incubator to allow the remaining parasites to continue growing
Detection and Characterization of Leptospira Infection and Exposure in Rats on the Caribbean Island of Saint Kitts
Full text linkIn this study, we detected and characterized Leptospira infection and exposure in rats on the Caribbean island of Saint Kitts for the first time. We detected Leptospira infection in 17/29 (59%), 14/29 (48) %, and 11/29 (38) % of rats by RT-PCR, culture, and immunofluorescence assay, respectively. Whole genome sequencing (WGS) and analysis and serogrouping of 17 Leptospira strains isolated from rats revealed their close relationship with L. interrogans serogroup Icterohaemorrhagiae (n = 10) and L. borgpetersenii serogroup Ballum (n = 7). WGS, serogrouping, and additional PCR tests on rat kidneys confirmed mixed infections with L. interrogans and L. borgpetersenii in the kidneys of three rats. Microscopic agglutination test (MAT) was positive for 25/29 (87%) of the rats tested, and the response was restricted to serovars Icterohaemorrhagiae {24/29(83%)}, Mankarso {4/29(14%)}, Copenhageni {4/29(14%)}, Grippotyphosa {2/29(7%)}, and Wolffi {1/29(3%)}. Interestingly, there was no agglutinating antibody response to serovar Ballum. We observed a similar pattern in the serologic response using Leptospira isolates obtained from this study with each of the rat sera, with strong response to L. interrogans isolates but minimal reactivity to L. borgpetersenii isolates. Our findings suggest the use of multiple complementary diagnostic tests for Leptospira surveillance and diagnosis to improve the accuracy of the dataIn this study, we detected and characterized Leptospira infection and exposure in rats on the Caribbean island of Saint Kitts for the first time. We detected Leptospira infection in 17/29 (59%), 14/29 (48) %, and 11/29 (38) % of rats by RT-PCR, culture, and immunofluorescence assay, respectively. Whole genome sequencing (WGS) and analysis and serogrouping of 17 Leptospira strains isolated from rats revealed their close relationship with L. interrogans serogroup Icterohaemorrhagiae (n = 10) and L. borgpetersenii serogroup Ballum (n = 7). WGS, serogrouping, and additional PCR tests on rat kidneys confirmed mixed infections with L. interrogans and L. borgpetersenii in the kidneys of three rats. Microscopic agglutination test (MAT) was positive for 25/29 (87%) of the rats tested, and the response was restricted to serovars Icterohaemorrhagiae {24/29(83%)}, Mankarso {4/29(14%)}, Copenhageni {4/29(14%)}, Grippotyphosa {2/29(7%)}, and Wolffi {1/29(3%)}. Interestingly, there was no agglutinating antibody response to serovar Ballum. We observed a similar pattern in the serologic response using Leptospira isolates obtained from this study with each of the rat sera, with strong response to L. interrogans isolates but minimal reactivity to L. borgpetersenii isolates. Our findings suggest the use of multiple complementary diagnostic tests for Leptospira surveillance and diagnosis to improve the accuracy of the dat
Mapping geographical inequalities in access to drinking water and sanitation facilities in low-income and middle-income countries, 2000–17
Full text linkBackground Universal access to safe drinking water and sanitation facilities is an essential human right, recognised in the Sustainable Development Goals as crucial for preventing disease and improving human wellbeing. Comprehensive, high-resolution estimates are important to inform progress towards achieving this goal. We aimed to produce high-resolution geospatial estimates of access to drinking water and sanitation facilities.
Methods We used a Bayesian geostatistical model and data from 600 sources across more than 88 low-income and middle-income countries (LMICs) to estimate access to drinking water and sanitation facilities on continuous continent-wide surfaces from 2000 to 2017, and aggregated results to policy-relevant administrative units. We estimated mutually exclusive and collectively exhaustive subcategories of facilities for drinking water (piped water on or off premises, other improved facilities, unimproved, and surface water) and sanitation facilities (septic or sewer sanitation, other improved, unimproved, and open defecation) with use of ordinal regression. We also estimated the number of diarrhoeal deaths in children younger than 5 years attributed to unsafe facilities and estimated deaths that were averted by increased access to safe facilities in 2017, and analysed geographical inequality in access within LMICs.
Findings Across LMICs, access to both piped water and improved water overall increased between 2000 and 2017, with progress varying spatially. For piped water, the safest water facility type, access increased from 40·0% (95% uncertainty interval [UI] 39·4–40·7) to 50·3% (50·0–50·5), but was lowest in sub-Saharan Africa, where access to piped water was mostly concentrated in urban centres. Access to both sewer or septic sanitation and improved sanitation overall also increased across all LMICs during the study period. For sewer or septic sanitation, access was 46·3% (95% UI 46·1–46·5) in 2017, compared with 28·7% (28·5–29·0) in 2000. Although some units improved access to the safest drinking water or sanitation facilities since 2000, a large absolute number of people continued to not have access in several units with high access to such facilities (>80%) in 2017. More than 253 000 people did not have access to sewer or septic sanitation facilities in the city of Harare, Zimbabwe, despite 88·6% (95% UI 87·2–89·7) access overall. Many units were able to transition from the least safe facilities in 2000 to safe facilities by 2017; for units in which populations primarily practised open defecation in 2000, 686 (95% UI 664–711) of the 1830 (1797–1863) units transitioned to the use of improved sanitation. Geographical disparities in access to improved water across units decreased in 76·1% (95% UI 71·6–80·7) of countries from 2000 to 2017, and in 53·9% (50·6–59·6) of countries for access to improved sanitation, but remained evident subnationally in most countries in 2017.
Interpretation Our estimates, combined with geospatial trends in diarrhoeal burden, identify where efforts to increase access to safe drinking water and sanitation facilities are most needed. By highlighting areas with successful approaches or in need of targeted interventions, our estimates can enable precision public health to effectively progress towards universal access to safe water and sanitation.Background Universal access to safe drinking water and sanitation facilities is an essential human right, recognised in the Sustainable Development Goals as crucial for preventing disease and improving human wellbeing. Comprehensive, high-resolution estimates are important to inform progress towards achieving this goal. We aimed to produce high-resolution geospatial estimates of access to drinking water and sanitation facilities.
Methods We used a Bayesian geostatistical model and data from 600 sources across more than 88 low-income and middle-income countries (LMICs) to estimate access to drinking water and sanitation facilities on continuous continent-wide surfaces from 2000 to 2017, and aggregated results to policy-relevant administrative units. We estimated mutually exclusive and collectively exhaustive subcategories of facilities for drinking water (piped water on or off premises, other improved facilities, unimproved, and surface water) and sanitation facilities (septic or sewer sanitation, other improved, unimproved, and open defecation) with use of ordinal regression. We also estimated the number of diarrhoeal deaths in children younger than 5 years attributed to unsafe facilities and estimated deaths that were averted by increased access to safe facilities in 2017, and analysed geographical inequality in access within LMICs.
Findings Across LMICs, access to both piped water and improved water overall increased between 2000 and 2017, with progress varying spatially. For piped water, the safest water facility type, access increased from 40·0% (95% uncertainty interval [UI] 39·4–40·7) to 50·3% (50·0–50·5), but was lowest in sub-Saharan Africa, where access to piped water was mostly concentrated in urban centres. Access to both sewer or septic sanitation and improved sanitation overall also increased across all LMICs during the study period. For sewer or septic sanitation, access was 46·3% (95% UI 46·1–46·5) in 2017, compared with 28·7% (28·5–29·0) in 2000. Although some units improved access to the safest drinking water or sanitation facilities since 2000, a large absolute number of people continued to not have access in several units with high access to such facilities (>80%) in 2017. More than 253 000 people did not have access to sewer or septic sanitation facilities in the city of Harare, Zimbabwe, despite 88·6% (95% UI 87·2–89·7) access overall. Many units were able to transition from the least safe facilities in 2000 to safe facilities by 2017; for units in which populations primarily practised open defecation in 2000, 686 (95% UI 664–711) of the 1830 (1797–1863) units transitioned to the use of improved sanitation. Geographical disparities in access to improved water across units decreased in 76·1% (95% UI 71·6–80·7) of countries from 2000 to 2017, and in 53·9% (50·6–59·6) of countries for access to improved sanitation, but remained evident subnationally in most countries in 2017.
Interpretation Our estimates, combined with geospatial trends in diarrhoeal burden, identify where efforts to increase access to safe drinking water and sanitation facilities are most needed. By highlighting areas with successful approaches or in need of targeted interventions, our estimates can enable precision public health to effectively progress towards universal access to safe water and sanitation
A multi-faceted genotoxic network of alpha-synuclein in the nucleus and mitochondria of dopaminergic neurons in Parkinson’s disease: Emerging concepts and challenges
No full textα-Synuclein is a hallmark amyloidogenic protein component of the Lewy bodies (LBs) present in dopaminergic neurons affected by Parkinson’s disease (PD). Despite an enormous increase in emerging knowledge, the mechanism(s) of α-synuclein neurobiology and crosstalk among pathological events that are critical for PD progression remains enigmatic, creating a roadblock for effective intervention strategies. One confounding question is about the potential link between α-synuclein toxicity and genome instability in PD. We previously reported that pro-oxidant metal ions, together with reactive oxygen species (ROS), act as a “double whammy” in dopaminergic neurons by not only inducing genome damage but also inhibiting their repair. Our recent studies identified a direct role for chromatin-bound, oxidized α-synuclein in the induction of DNA strand breaks, which raised the question of a paradoxical role for α-synuclein’s DNA binding in neuroprotection versus neurotoxicity. Furthermore, recent advances in our understanding of α-synuclein mediated mitochondrial dysfunction warrants revisiting the topics of α-synuclein pathophysiology in order to devise and assess the efficacy of α-synuclein-targeted interventions. In this review article, we discuss the multi-faceted neurotoxic role of α-synuclein in the nucleus and mitochondria with a particular emphasis on the role of α-synuclein in DNA damage/repair defects. We utilized a protein-DNA binding simulation to identify potential residues in α-synuclein that could mediate its binding to DNA and may be critical for its genotoxic functions. These emerging insights and paradigms may guide new drug targets and therapeutic modalities.α-Synuclein is a hallmark amyloidogenic protein component of the Lewy bodies (LBs) present in dopaminergic neurons affected by Parkinson’s disease (PD). Despite an enormous increase in emerging knowledge, the mechanism(s) of α-synuclein neurobiology and crosstalk among pathological events that are critical for PD progression remains enigmatic, creating a roadblock for effective intervention strategies. One confounding question is about the potential link between α-synuclein toxicity and genome instability in PD. We previously reported that pro-oxidant metal ions, together with reactive oxygen species (ROS), act as a “double whammy” in dopaminergic neurons by not only inducing genome damage but also inhibiting their repair. Our recent studies identified a direct role for chromatin-bound, oxidized α-synuclein in the induction of DNA strand breaks, which raised the question of a paradoxical role for α-synuclein’s DNA binding in neuroprotection versus neurotoxicity. Furthermore, recent advances in our understanding of α-synuclein mediated mitochondrial dysfunction warrants revisiting the topics of α-synuclein pathophysiology in order to devise and assess the efficacy of α-synuclein-targeted interventions. In this review article, we discuss the multi-faceted neurotoxic role of α-synuclein in the nucleus and mitochondria with a particular emphasis on the role of α-synuclein in DNA damage/repair defects. We utilized a protein-DNA binding simulation to identify potential residues in α-synuclein that could mediate its binding to DNA and may be critical for its genotoxic functions. These emerging insights and paradigms may guide new drug targets and therapeutic modalities
COVID-19 in Latin America: Novel transmission dynamics for a global pandemic?
Full text linkThe COVID-19 virus expanded from China into Western Asia, Europe, and North America, impacting many of the world’s wealthiest countries. Brazil reported Latin America’s first case in late February 2020, and in less than a month, over 7,000 COVID-19 cases have been confirmed among nearly every country and territory in Latin America and the Caribbean (LAC). The LAC outbreak appears to be about two weeks behind the United States and Canada and about three to four weeks behind Western Europe. Thus, the global COVID-19 pandemic is entering a new phase, not only expanding beyond primarily temperate Northern Hemisphere countries into the tropics but also spreading to a geopolitical region marked by significantly worse poverty, water access and sanitation, and distrust in public governance (Fig 1). We believe that these aspects of the Latin American context are likely to substantially affect the transmission dynamics and scope of the COVID-19 outbreak in LAC, with potential implications for the trajectory of the global pandemic.The COVID-19 virus expanded from China into Western Asia, Europe, and North America, impacting many of the world’s wealthiest countries. Brazil reported Latin America’s first case in late February 2020, and in less than a month, over 7,000 COVID-19 cases have been confirmed among nearly every country and territory in Latin America and the Caribbean (LAC). The LAC outbreak appears to be about two weeks behind the United States and Canada and about three to four weeks behind Western Europe. Thus, the global COVID-19 pandemic is entering a new phase, not only expanding beyond primarily temperate Northern Hemisphere countries into the tropics but also spreading to a geopolitical region marked by significantly worse poverty, water access and sanitation, and distrust in public governance (Fig 1). We believe that these aspects of the Latin American context are likely to substantially affect the transmission dynamics and scope of the COVID-19 outbreak in LAC, with potential implications for the trajectory of the global pandemic
Betacoronavirus genomes: How genomic information has been used to deal with past outbreaks and the COVID-19 pandemic
Full text linkIn the 21st century, three highly pathogenic betacoronaviruses have emerged, with an alarming rate of human morbidity and case fatality. Genomic information has been widely used to understand the pathogenesis, animal origin and mode of transmission of betacoronaviruses in the aftermath of the 2002-03 severe acute respiratory syndrome (SARS) and 2012 Middle East respiratory syndrome (MERS) outbreaks. Furthermore, genome sequencing and bioinformatic analysis have had an unprecedented relevance in the battle against the 2019-20 coronavirus disease 2019 (COVID-19) pandemic, the newest and most devastating outbreak caused by a coronavirus in the history of mankind, allowing the follow up of disease spread and transmission dynamics in near real time. Here, we review how genomic information has been used to tackle outbreaks caused by emerging, highly pathogenic, betacoronavirus strains, emphasizing on SARS-CoV, MERS-CoV and SARS-CoV-2.In the 21st century, three highly pathogenic betacoronaviruses have emerged, with an alarming rate of human morbidity and case fatality. Genomic information has been widely used to understand the pathogenesis, animal origin and mode of transmission of betacoronaviruses in the aftermath of the 2002-03 severe acute respiratory syndrome (SARS) and 2012 Middle East respiratory syndrome (MERS) outbreaks. Furthermore, genome sequencing and bioinformatic analysis have had an unprecedented relevance in the battle against the 2019-20 coronavirus disease 2019 (COVID-19) pandemic, the newest and most devastating outbreak caused by a coronavirus in the history of mankind, allowing the follow up of disease spread and transmission dynamics in near real time. Here, we review how genomic information has been used to tackle outbreaks caused by emerging, highly pathogenic, betacoronavirus strains, emphasizing on SARS-CoV, MERS-CoV and SARS-CoV-2
First Whole Genome Sequence of Anaplasma platys, an Obligate Intracellula Rickettsial Pathogen of Dogs
Full text linkWe have assembled the first genome draft of Anaplasma platys, an obligate intracellular rickettsia, and the only known bacterial pathogen infecting canine platelets. A. platys is a not-yet-cultivated bacterium that causes infectious cyclic canine thrombocytopenia, a potentially fatal disease in dogs. Despite its global distribution and veterinary relevance, no genome sequence has been published so far for this pathogen. Here, we used a strategy based on metagenome assembly to generate a draft of the A. platys genome using the blood of an infected dog. The assembled draft is similar to other Anaplasma genomes in size, gene content, and synteny. Notable differences are the apparent absence of rbfA, a gene encoding a 30S ribosome-binding factor acting as a cold-shock protein, as well as two genes involved in biotin metabolism. We also observed differences associated with expanded gene families, including those encoding outer membrane proteins, a type IV secretion system, ankyrin repeat-containing proteins, and proteins with predicted intrinsically disordered regions. Several of these families have members highly divergent in sequence, likely to be associated with survival and interactions within the host and the vector. The sequence of the A. platys genome can benefit future studies regarding invasion, survival, and pathogenesis of Anaplasma species, while paving the way for the better design of treatment and prevention strategies against these neglected intracellular pathogens.We have assembled the first genome draft of Anaplasma platys, an obligate intracellular rickettsia, and the only known bacterial pathogen infecting canine platelets. A. platys is a not-yet-cultivated bacterium that causes infectious cyclic canine thrombocytopenia, a potentially fatal disease in dogs. Despite its global distribution and veterinary relevance, no genome sequence has been published so far for this pathogen. Here, we used a strategy based on metagenome assembly to generate a draft of the A. platys genome using the blood of an infected dog. The assembled draft is similar to other Anaplasma genomes in size, gene content, and synteny. Notable differences are the apparent absence of rbfA, a gene encoding a 30S ribosome-binding factor acting as a cold-shock protein, as well as two genes involved in biotin metabolism. We also observed differences associated with expanded gene families, including those encoding outer membrane proteins, a type IV secretion system, ankyrin repeat-containing proteins, and proteins with predicted intrinsically disordered regions. Several of these families have members highly divergent in sequence, likely to be associated with survival and interactions within the host and the vector. The sequence of the A. platys genome can benefit future studies regarding invasion, survival, and pathogenesis of Anaplasma species, while paving the way for the better design of treatment and prevention strategies against these neglected intracellular pathogens
COVID-19 pandemic in Panama: lessons of the unique risks and research opportunities for Latin America
Full text linkThe Republic of Panama has the second most unequally distributed wealth in Central America, has recently entered the list of countries affected by the COVID-19 pandemic, and has one of the largest testing rate per inhabitant in the region and consequently the highest incidence rate of COVID-19, making it an ideal location to discuss potential scenarios for assessing epidemic preparedness, and to outline research opportunities in the Region of the Americas. We address two timely important questions: What are the unique risks of COVID-19 in Panama that could help other countries in the Region be better prepared? And what kind of scientific knowledge can Panama contribute to the regional and global study of COVID-19? This paper provides suggestions about how the research community could support local health authorities plan for different scenarios and decrease public anxiety. It also presents basic scientific opportunities about emerging pandemic pathogens towards promoting global health from the perspective of a middle income countryThe Republic of Panama has the second most unequally distributed wealth in Central America, has recently entered the list of countries affected by the COVID-19 pandemic, and has one of the largest testing rate per inhabitant in the region and consequently the highest incidence rate of COVID-19, making it an ideal location to discuss potential scenarios for assessing epidemic preparedness, and to outline research opportunities in the Region of the Americas. We address two timely important questions: What are the unique risks of COVID-19 in Panama that could help other countries in the Region be better prepared? And what kind of scientific knowledge can Panama contribute to the regional and global study of COVID-19? This paper provides suggestions about how the research community could support local health authorities plan for different scenarios and decrease public anxiety. It also presents basic scientific opportunities about emerging pandemic pathogens towards promoting global health from the perspective of a middle income countr
Complete Genome Sequence of a Virulent Leptospira interrogans Serovar Copenhageni Strain, Assembled with a Combination of Nanopore and Illumina Reads
Full text linkHere, we present the complete genome sequence of a highly virulent Leptospira interrogans serovar Copenhageni strain isolated from a dog with severe leptospirosis. In this work, a gapless genome draft was assembled with a combination of Nanopore and Illumina data of relatively low coverage.Here, we present the complete genome sequence of a highly virulent Leptospira interrogans serovar Copenhageni strain isolated from a dog with severe leptospirosis. In this work, a gapless genome draft was assembled with a combination of Nanopore and Illumina data of relatively low coverage
Agua Salud alphavirus defines a novel lineage of insect- specific alphaviruses discovered in the New World
Full text linkThe genus Alphavirus harbours mostly insect- transmitted viruses that cause severe disease in humans, livestock and wildlife. Thus far, only three alphaviruses with a host range restricted to insects have been found in mosquitoes from the Old World, namely Eilat virus (EILV), Taï Forest alphavirus (TALV) and Mwinilunga alphavirus (MWAV). In this study, we found a novel alphavirus in one Culex declarator mosquito sampled in Panama. The virus was isolated in C6/36 mosquito cells, and full genome sequencing revealed an 11 468 nt long genome with maximum pairwise nucleotide identity of 62.7 % to Sindbis virus. Phylogenetic analyses placed the virus as a solitary deep rooting lineage in a basal relationship to the Western equine encephalitis antigenic complex and to the clade comprising EILV, TALV and MWAV, indicating the detection of a novel alphavirus, tentatively named Agua Salud alphavirus (ASALV). No growth of ASALV was detected in vertebrate cell lines, including cell lines derived from ectothermic animals, and replication of ASALV was strongly impaired above 31 °C, suggesting that ASALV represents the first insect- restricted alphavirus of the New World.The genus Alphavirus harbours mostly insect- transmitted viruses that cause severe disease in humans, livestock and wildlife. Thus far, only three alphaviruses with a host range restricted to insects have been found in mosquitoes from the Old World, namely Eilat virus (EILV), Taï Forest alphavirus (TALV) and Mwinilunga alphavirus (MWAV). In this study, we found a novel alphavirus in one Culex declarator mosquito sampled in Panama. The virus was isolated in C6/36 mosquito cells, and full genome sequencing revealed an 11 468 nt long genome with maximum pairwise nucleotide identity of 62.7 % to Sindbis virus. Phylogenetic analyses placed the virus as a solitary deep rooting lineage in a basal relationship to the Western equine encephalitis antigenic complex and to the clade comprising EILV, TALV and MWAV, indicating the detection of a novel alphavirus, tentatively named Agua Salud alphavirus (ASALV). No growth of ASALV was detected in vertebrate cell lines, including cell lines derived from ectothermic animals, and replication of ASALV was strongly impaired above 31 °C, suggesting that ASALV represents the first insect- restricted alphavirus of the New World