Clinical and epidemiological characteristics of Madariaga and Venezuelan equine encephalitis virus infections.

Madariaga virus (MADV) and Venezuelan equine encephalitis virus (VEEV) are emerging arboviruses affecting rural and remote areas of Latin America. However, there are limited clinical and epidemiological reports available, and outbreaks are occurring at an increasing frequency. We addressed this gap by analyzing all the available clinical and epidemiological data of MADV and VEEV infections recorded since 1961 in Panama. A total of 168 of human alphavirus encephalitis cases were detected in Panama from 1961 to 2023. Here we describe the clinical signs and symptoms and epidemiological characteristics of these cases, and also explored signs and symptoms as potential predictors of encephalitic alphavirus infection when compared to those of other arbovirus infections occurring in the region. Our results highlight the challenges clinical diagnosis of alphavirus disease in endemic regions with overlapping circulation of multiple arboviruses.

Madariaga and Venezuelan equine encephalitis virus seroprevalence in rodent enzootic hosts in Eastern and Western Panama.

While rodents are primary reservoirs of Venezuelan equine encephalitis virus (VEEV), their role in Madariaga virus (MADV) transmission remains uncertain, particularly given their overlapping geographic distribution. This study explores the interplay of alphavirus prevalence, rodent diversity, and land use within Darien and Western Panama provinces. A total of three locations were selected for rodent sampling in Darien province: Los Pavitos, El Real de Santa Maria and Santa Librada. Two sites were selected in Western Panama province: El Cacao and Cirí Grande. We used plaque reduction neutralization tests to assess MADV and VEEV seroprevalences in 599 rodents of 16 species across five study sites. MADV seroprevalence was observed at higher rates in Los Pavitos (Darien province), 9.0%, 95% CI: 3.6-17.6, while VEEV seroprevalence was elevated in El Cacao (Western Panama province), 27.3%, 95% CI: 16.1-40.9, and El Real de Santa María (Darien province), 20.4%, 95% CI: 12.6-29.7. Species like Oryzomys coesi, 23.1%, 95% CI: 5.0-53.8, and Transandinomys bolivaris, 20.0%, 95% CI: 0.5-71.6 displayed higher MADV seroprevalences than other species, whereas Transandinomys bolivaris, 80.0%, 95% CI: 28.3-99.4, and Proechimys semispinosus, 27.3%, 95% CI: 17.0-39.6, exhibited higher VEEV seroprevalences. Our findings provide support to the notion that rodents are vertebrate reservoirs of MADV and reveal spatial variations in alphavirus seropositivity among rodent species, with different provinces exhibiting distinct rates for MADV and VEEV. Moreover, specific rodent species are linked to unique seroprevalence patterns for these viruses, suggesting that rodent diversity and environmental conditions might play a significant role in shaping alphavirus distribution.

Phylogenetic characterization of Orthobunyaviruses isolated from Trinidad shows evidence of natural reassortment.

The genus Orthobunyavirus is a diverse group of viruses in the family Peribunyaviridae, recently classified into 20 serogroups, and 103 virus species. Although most viruses within these serogroups are phylogenetically distinct, the absence of complete genome sequences has left several viruses incompletely characterized. Here we report the complete genome sequences for 11 orthobunyaviruses isolated from Trinidad, French Guiana, Guatemala, and Panama that were serologically classified into six serogroups and 10 species. Phylogenetic analyses of these 11 newly derived sequences indicate that viruses belonging to the Patois, Capim, Guama, and Group C serocomplexes all have a close genetic origin. We show that three of the 11 orthobunyaviruses characterized (belonging to the Group C and Bunyamwera serogroups) have evidence of histories of natural reassortment through the M genome segment. Our data also suggests that two distinct lineages of Group C viruses concurrently circulate in Trinidad and are transmitted by the same mosquito vectors. This study also highlights the importance of complementing serological identification with nucleotide sequencing when characterizing orthobunyaviruses.

Etiologies of Acute Undifferentiated Febrile Illnesses in and near Iquitos from 1993 to 1999 in the Amazon River Basin of Peru.

The objective of this study was to determine the etiology of febrile illnesses among patients from October 1, 1993 through September 30, 1999, in the urban community of Iquitos in the Amazon River Basin of Peru. Epidemiological and clinical data as well as blood samples were obtained from consenting patients at hospitals, health clinics and private residences. Samples were tested for arboviruses in cell cultures and for IgM and IgG antibodies by ELISA. Blood smears were examined for malaria, and sera were tested for antibodies to Leptospira spp. by ELISA and microscopic agglutination. Among 6,607 febrile patients studied, dengue viruses caused 14.6% of the cases, and Venezuelan equine encephalitis virus caused 2.5%, Oropouche virus 1.0%, Mayaro virus 0.4%, and other arboviruses caused 0.2% of the cases. Also, 22.9% of 4,844 patients tested were positive for malaria, and of 400 samples tested, 9% had evidence of acute leptospirosis. Although the study was not designed to assess the importance of these pathogens as a cause of human morbidity in the total population, these results indicate that arboviruses, leptospirosis, and malaria were the cause of approximately 50% of the febrile cases. Although the arboviruses that were diagnosed can produce asymptomatic infections, our findings increased the overall understanding of the relative health burden of these infections, as well as baseline knowledge needed for designing and implementing further studies to better assess the health impact and threat of these pathogens in the Amazon Basin of Peru.

Genomic characterization of Changuinola viruses from Panama: evidence for multiple genome segment reassortment.

The complete coding sequences of five divergent strains of Changuinola virus (CGLV), collected over a 16-year period in Panama, were determined, using viral metagenomics. Each strain had 10 RNA segments that encoded structural and non-structural proteins with amino acid identities ranging from 33 to 99% with sequences of other 15 members of the Changuinola virus (Reoviridae: Orbivirus) species group. Genetic analyses of the five Panamanian virus strains revealed probable reassortment among multiple segments of the viruses.

Characterization of Triniti virus supports its reclassification in the family Peribunyaviridae.

Triniti virus (TNTV) has been isolated in Trinidad and Tobago and in Brazil. To date little is known about this virus, which is classified as an ungrouped virus within the family Togaviridae. Here, three isolates of TNTV were characterized both genetically and antigenically. The genome was shown to contain three RNA segments: small (S), medium (M) and large (L). Genome organization, protein sizes and protein motifs were similar to those of viruses in the genus Orthobunyavirus, family Peribunyaviridae. Antigenic reactivity revealed the three TNTV isolates to be closely related, but no serologic cross-reaction with other orthobunyaviruses. Morphological observation by transmission electron microscopy indicated that virus size and symmetry were compatible with those of viruses in the family Peribunyaviridae. Our serological, morphological and molecular results support the taxonomic reclassification of TNTV as a member of the genus Orthobunyavirus, family Peribunyaviridae.

Genome Sequence of Chiqui Virus, a Novel Reovirus Isolated from Mosquitoes Collected in Colombia.

We report here the complete genome sequence of a novel reovirus, designated Chiqui virus (CHQV) strain CoB38d, that was isolated from a pool of unidentified mosquitoes collected in northern Colombia in 2013. CHQV has nine double-stranded DNA (dsRNA) genome segments and has similarity to viruses belonging to the family Reoviridae, subfamily Spinareovirinae.

Characterization of the Gamboa Virus Serogroup (Orthobunyavirus Genus, Peribunyaviridae Family).

Comprehensive comparative phylogenetic analyses were performed on 17 Gamboa serogroup viruses (GAMSVs) from distinct geographic regions in the Americas and other representative members of the genus Orthobunyavirus (Peribunyaviridae), based on small (S), medium (M), and large (L) open reading frame full-length and partial sequences. Genome characterization showed that the GAMSVs divide into four clades or genotypes. The GAMSVs have a genetic organization similar to other orthobunyaviruses, except that they have a larger NSm protein than other orthobunyaviruses. A serosurvey for Gamboa virus antibodies was performed in plasma from birds, other wild animals, and humans living around the Tucuruí hydroelectric dam in Pará state, northern Brazil, a known focus of GAMSV activity. Newborn chicks (Gallus gallus domesticus) were experimentally infected with a GAMSV, and the pathogenesis is described. Histopathological changes were primarily in the lungs and liver. Also, a review of the ecology of the GAMSVs in the Americas is included. In sum, this study presents the genomic and evolutionary characterization of the Gamboa group and the potential model of pathogenesis, which would be helpful for diagnostic purposes, epidemiology, and immunopathogenesis studies.

The reintroduction of DENV-2 in 2011 in Panama and subsequent outbreak characteristic.

The circulation of the South-east Asian/American (AS/AM) dengue 2 virus (DENV-2) genotype in the Americas has been associated with a high rate of severe disease. From 1993, the year DENV was reintroduced in Panama, until 2011 there were 29 dengue-associated deaths, 17 of which occurred in 2011, the most severe outbreak with a case fatality rate (CFR) of 44% (17 deaths out of 38 severe dengue cases). During this outbreak DENV-2 was reintroduced into the country, whereas over the prior five years DENV-1 and -3 were predominant. Herein, we describe the 2011 Panama outbreak and genetically characterize the Panamanian DENV-2 strains, which were associated with severe dengue disease in Panama. Our results suggest that the DENV-2 isolates from this outbreak belonged to the AS/AM genotype sub-clade 2BI and were genetically close to viruses described in the outbreaks in Nicaragua, Honduras, Guatemala and Mexico from 2006-2011. Sub-clade 2BI has previously been associated with severe disease in Nicaragua during outbreaks from 2005-2007.

Characterization of Three New Insect-Specific Flaviviruses: Their Relationship to the Mosquito-Borne Flavivirus Pathogens.

Three novel insect-specific flaviviruses, isolated from mosquitoes collected in Peru, Malaysia (Sarawak), and the United States, are characterized. The new viruses, designated La Tina, Kampung Karu, and Long Pine Key, respectively, are antigenically and phylogenetically more similar to the mosquito-borne flavivirus pathogens, than to the classical insect-specific viruses like cell fusing agent and Culex flavivirus. The potential implications of this relationship and the possible uses of these and other arbovirus-related insect-specific flaviviruses are reviewed.

Experimental Infection with and Maintenance of Cell Fusing Agent Virus (Flavivirus) in Aedes aegypti.

During the past two decades, there has been a dramatic increase in the recognition and characterization of novel insect-specific flaviviruses (ISFVs). Some of these agents are closely related to important mosquito-borne flavivirus pathogens. Results of experimental studies suggest that mosquitoes and mosquito cell cultures infected with some ISFVs are refractory to superinfection with related flavivirus pathogens; and it has been proposed that ISFVs potentially could be used to alter the vector competence of mosquitoes and reduce transmission of specific flavivirus pathogens, such as dengue, West Nile, or Zika viruses. In order for an ISFV to be used in such a control strategy, the virus would have to be vertically transmitted at a high rate in the target vector population to insure its continued maintenance. This study compared the vertical transmission rates of an ISFV, cell fusing agent virus (CFAV), in two Aedes aegypti colonies: one naturally infected with CFAV and the other experimentally infected but previously free of the virus. CFAV filial infection rates in progeny of female mosquitoes from both colonies were > 90% after two generations of selection, indicating the feasibility of introducing an ISFV into a mosquito population. This and other considerations for evaluating the feasibility of using ISFVs as an arbovirus control strategy are discussed.

Genetic characterization, molecular epidemiology, and phylogenetic relationships of insect-specific viruses in the taxon Negevirus.

The recently described taxon Negevirus is comprised of a diverse group of insect-specific viruses isolated from mosquitoes and phlebotomine sandflies. In this study, a comprehensive genetic characterization, molecular, epidemiological and evolutionary analyses were conducted on nearly full-length sequences of 91 new negevirus isolates obtained in Brazil, Colombia, Peru, Panama, USA and Nepal. We demonstrated that these arthropod restricted viruses are clustered in two major phylogenetic groups with origins related to three plant virus genera (Cilevirus, Higrevirus and Blunevirus). Molecular analyses demonstrated that specific host correlations are not present with most negeviruses; instead, high genetic variability, wide host-range, and cross-species transmission were noted. The data presented here also revealed the existence of five novel insect-specific viruses falling into two arthropod-restrictive virus taxa, previously proposed as distinct genera, designated Nelorpivirus and Sandewavirus. Our results provide a better understanding of the molecular epidemiology, evolution, taxonomy and stability of this group of insect-restricted viruses.

Almendravirus: A Proposed New Genus of Rhabdoviruses Isolated from Mosquitoes in Tropical Regions of the Americas.

The Rhabdoviridae is a diverse family of negative-sense single-stranded RNA viruses, many of which infect vertebrate hosts and are transmitted by hematophagous arthropods. Others appear to be arthropod specific, circulating only within arthropod populations. Herein, we report the isolation and characterization of three novel viruses from mosquitoes collected from the Americas. Coot Bay virus was isolated from Anopheles quadrimaculatus mosquitoes collected in the Everglades National Park, Florida; Rio Chico virus was isolated from Anopheles triannulatus mosquitoes collected in Panama; and Balsa virus was isolated from two pools of Culex erraticus mosquitoes collected in Colombia. Sequence analysis indicated that the viruses share a similar genome organization to Arboretum virus and Puerto Almendras virus that had previously been isolated from mosquitoes collected in Peru. Each genome features the five canonical rhabdovirus structural protein genes as well as a gene encoding a class 1A viroporin-like protein (U1) located between the G and L genes (3'-N-P-M-G-U1-L-5'). Phylogenetic analysis of complete L protein sequences indicated that all five viruses cluster in a unique clade that is relatively deeply rooted in the ancestry of animal rhabdoviruses. The failure of all viruses in this clade to grow in newborn mice or vertebrate cells in culture suggests that they may be poorly adapted to replication in vertebrates.

Sinu virus, a novel and divergent orthomyxovirus related to members of the genus Thogotovirus isolated from mosquitoes in Colombia.

The genome and structural organization of a novel insect-specific orthomyxovirus, designated Sinu virus, is described. Sinu virus (SINUV) was isolated in cultures of C6/36 cells from a pool of mosquitoes collected in northwestern Colombia. The virus has six negative-sense ssRNA segments. Genetic analysis of each segment demonstrated the presence of six distinct ORFs encoding the following genes: PB2 (Segment 1), PB1, (Segment 2), PA protein (Segment 3), envelope GP gene (Segment 4), the NP (Segment 5), and M-like gene (Segment 6). Phylogenetically, SINUV appears to be most closed related to viruses in the genus Thogotovirus.

Genetic Characterization of Northwestern Colombian Chikungunya Virus Strains from the 2014-2015 Epidemic.

Chikungunya fever, an acute and often chronic arthralgic disease caused by the mosquito-borne alphavirus, chikungunya virus (CHIKV), spread into the Americas in late 2013. Since then it has caused epidemics in nearly all New World countries, the second largest being Colombia with over 450,000 suspected cases beginning in September, 2014, and focused in Bolivar Department in the north. We examined 32 human sera from suspected cases, including diverse age groups and both genders, and sequenced the CHIKV envelope glycoprotein genes, known determinants of vector host range. As expected for Asian lineage CHIKV strains, these isolates lacked known Aedes albopictus-adaptive mutations. All the Colombian strains were closely related to those from the Virgin Islands, Saint Lucia, Mexico, Puerto Rico, and Brazil, consistent with a single, point-source introduction from the southeast Asia/Pacific region. Two substitutions in the E2 and E1 envelope glycoprotein genes were found in the Colombian strains, especially E1-K211E involving a residue shown previously to affect epistatically the penetrance of the E1-A226V A. albopictus-adaptive substitution. We also identified two amino acid substitutions unique to all American CHIKV sequences: E2-V368A and 6K-L20M. Only one codon, 6K-47, had a high nonsynonymous substitution rate suggesting positive selection.

Analysis of a Reverse Transcription Loop-mediated Isothermal Amplification (RT-LAMP) for yellow fever diagnostic.

Yellow Fever virus (YFV) is an important human pathogen in tropical areas of Africa and South America. Although an efficient vaccine is available and has been used since the early 1940s, sylvatic YFV transmission still occurs in forested areas where anthropogenic actions are present, such as mineral extraction, rearing livestock and agriculture, and ecological tourism. In this context, two distinct techniques based on the RT-PCR derived method have been previously developed, however both methods are expensive due to the use of thermo cyclers and labeled probes. We developed isothermal genome amplification, which is a rapid, sensitive, specific and low cost molecular approach for YFV genome detection. This assay used a set of degenerate primers designed for the NS1 gene and was able to amplify, within 30 min in isothermal conditions, the YFV 17D vaccine strain derived from an African wild prototype strain (Asibi), as well as field strains from Brazil, other endemic countries from South and Central America, and the Caribbean. The generic RT-LAMP assay could be helpful for YFV surveillance in field and rapid response during outbreaks in endemic areas.

Mercadeo Virus: A Novel Mosquito-Specific Flavivirus from Panama.

Viruses in the genus Flavivirus (family Flaviviridae) include many arthropod-borne viruses of public health and veterinary importance. However, during the past two decades an explosion of novel insect-specific flaviviruses (ISFs), some closely related to vertebrate pathogens, have been discovered. Although many flavivirus pathogens of vertebrates have been isolated from naturally infected mosquitoes in Panama, ISFs have not previously been reported from the country. This report describes the isolation and characterization of a novel ISF, tentatively named Mercadeo virus (MECDV), obtained from Culex spp. mosquitoes collected in Panama. Two MECDV isolates were sequenced and cluster phylogenetically with cell-fusing agent virus (CFAV) and Nakiwogo virus (NAKV) to form a distinct lineage within the insect-specific group of flaviviruses.

Characterization of the Punta Toro species complex (genus Phlebovirus, family Bunyaviridae).

Punta Toro virus (PTV), a member of the PTV complex, is a relatively common causative agent of febrile illness in Panama that is often misdiagnosed as 'dengue' or 'influenza'. Currently, only two named members make up this species complex, PTV and Buenaventura virus (BUEV). Genomic and antigenic characterization of 17 members of the PTV complex, nine of which were isolated from human acute febrile illness cases, reveals that this species complex is composed of six distant viruses. We propose to add four additional new viruses, designated Leticia virus, Cocle virus, Campana virus and Capira virus.

Emergence of new insect-restrictive viruses in the Amazon region.

The complete genome was determined for 12 viruses isolated from 8 different pools of mosquitoes (Culex sp. and Psorophora ferox) collected at Brejeira farm, Canaan dos Carajas, Para state in northern Brazil. Eight of the viruses were distantly related to Piura virus, hereafter designated as Brejeira virus; the other 4 were similar to Wallerfield virus.