Retrospective molecular investigation of Mayaro and Oropouche viruses at the human-animal interface in West-central Brazil, 2016-2018.

Mayaro virus (MAYV, Togaviridae) and Oropouche orthobunyavirus (OROV, Peribunyaviridae) are emerging enzootic arboviruses in Latin America. Outbreaks of febrile illness associated with MAYV and OROV have been reported among humans mainly in the northern region of Brazil since the 1980s, and recent data suggest these viruses have circulated also in more populated areas of western Brazil. MAYV shares mosquito vectors with yellow fever virus and it has been historically detected during yellow fever epidemics. Aiming to investigate the transmission of OROV and MAYV at the human-animal interface during a yellow fever, chikungunya and Zika outbreaks in Brazil, we conducted a retrospective molecular investigation in 810 wild and domestic animals, 106 febrile patients, and 22.931 vectors collected from 2016 to 2018 in Cuiaba and Campo Grande metropolitan regions, western Brazil. All samples tested negative for OROV and MAYV RNA by RT-qPCR. Findings presented here suggest no active circulation of MAYV and OROV in the sampled hosts. Active surveillance and retrospective investigations are instrumental approaches for the detection of cryptic and subclinical activity of enzootic arboviruses and together serve as a warning system to implement appropriate actions to prevent outbreaks.

Host selection pattern and flavivirus screening of mosquitoes in a disturbed Colombian rainforest.

Studies on the feeding behavior of hematophagous insects, particularly those of medical importance, are relevant for tracking possible pathogen transmission routes and identifying biases in the choice of vertebrates. We evaluated host selection of blood-feeding mosquitoes in a disturbed forest in the Magdalena Medio valley in Colombia from March 2017 to April 2018, after the introduction of Zika virus to the Americas from the 2015-2016 outbreak. We estimated vertebrate diversity and collected blood-engorged female mosquitoes. Genomic DNA/RNA was extracted from the mosquito's abdomen for vertebrate host identification and pathogen detection. We performed conventional PCR and sequencing, using universal primers targeting vertebrate regions of the eukaryotic mitochondrial genome to determine bloodmeal host. Additionally, we tested for the presence of flaviviruses in all mosquito samples with RT-PCR. Based on the identity and quantity of detected bloodmeals, we performed mosquito-vertebrate interaction network analysis and estimated topology metrics. In total, we collected 292 engorged female mosquitoes representing 20 different species. Bloodmeal analyses identified 26 vertebrate species, the majority of which were mammals (N = 16; 61.5%). No flaviviruses of medical importance were detected from the samples. Although feeding patterns varied, network analyses showed a high degree of specialization by mosquitoes and revealed ecological and phylogenetic relationships among the host community. We conclude that host selection or preference by mosquitoes is species specific.

Bloodmeal Host Selection of Culex quinquefasciatus (Diptera: Culicidae) in Las Vegas, Nevada, United States.

St. Louis encephalitis virus (SLEV) and West Nile virus (WNV) have recently emerged in the southwestern United States. Surveillance for arboviruses in Las Vegas, NV, detected a surge of SLEV activity in the southern house mosquito (Culex quinquefasciatus Say) during 2016. To identify candidate avian amplifiers, we assessed the identification, viral infection, and immune status of vertebrate hosts for 195 blood-engorged Cx. quinquefasciatus mosquitoes collected in August and September 2016. Bloodmeals were identified from 164 engorged abdomens, representing 19 species of birds and three species of mammals. No SLEV or WNV viremia was detected, but one mosquito tested positive for Culex flavivirus. House finch (Haemorhous mexicanus) (Muller) was the most common bloodmeal, followed by domestic chicken (Gallus gallus) (Linnaeus), American robin (Turdus migratorius) L., house sparrow (Passer domesticus) (L.), great-tailed grackle (Quiscalus mexicanus) (Gmelin), northern mockingbird (Mimus polyglottos) (L.) and mourning dove (Zenaida macroura) (L.). SLEV-reactive antibodies were detected in six identified bloodmeals and WNV-reactive antibodies were detected in 33. House sparrow and house finch were the most likely hosts to show previous exposure to SLEV and WNV, respectively. Over-utilization by Cx. quinquefasciatus for bloodmeal hosts was observed primarily among robin, finch and sparrow, all species that roost communally. House finch stands out as a candidate important amplifier for both SLEV and WNV because of its preference by mosquito vectors, and high competence for closely related virus strains. While implicated in previous outbreaks as an important mosquito vector, Cx. quinquefasciatus feeds infrequently on mammals in Las Vegas, indicating a low risk for bridge transmission to humans.

Neutralizing antibodies for orthobunyaviruses in Pantanal, Brazil.

The Pantanal is a hotspot for arbovirus studies in South America. Various medically important flaviviruses and alphaviruses have been reported in domestic and wild animals in the region. To expand the knowledge of local arbovirus circulation, a serosurvey for 14 Brazilian orthobunyaviruses was conducted with equines, sheep and free-ranging caimans. Sera were tested for specific viral antibodies using plaque-reduction neutralization test (PRNT). Monotypic reactions were detected for Maguari, Xingu, Apeu, Guaroa, Murutucu, Oriboca, Oropouche and Nepuyo viruses. Despite the low titers for most of the orthobunyaviruses tested, the detection of monotypic reactions for eight orthobunyaviruses suggests the Pantanal as a region of great orthobunyavirus diversity. The present data, in conjunction with previous studies that detected a high diversity of other arboviruses, ratify the Pantanal as an important natural reservoir for sylvatic and medically important arboviruses in Brazil.

VectorTest™ West Nile Virus Antigen Assay in an Inhibition Platform as Field Screening Tool for Flavivirus Group-Specific Antibodies in Brazilian Equines.

Current methods for detecting Flavivirus antibodies are enzyme-linked immunosorbent assays (ELISAs) and neutralization tests, both of which require laboratories and trained staff. We evaluated the VectorTest™ West Nile Virus Antigen Assay in an inhibition platform (VecTest-inhibition assay [VIA]) as a simpler screening method for detecting antibodies for a variety of flaviviruses among a population of equines from Brazil. We found that the VIA is a field-deployable rapid method with 100% sensitivity and 64% specificity compared with blocking ELISA for the detection of group-specific Flavivirus antibodies in equine serum samples. The VIA is a potentially useful field test for rapid field-based Flavivirus antibody detection in equine serum samples.

Novel Viruses Isolated from Mosquitoes in Pantanal, Brazil.

Genomic sequences are described from five novel viruses and divergent strains of Brejeira and Guaico Culex viruses from mosquitoes collected in Pantanal, Brazil, in 2010.

Activity Patterns of St. Louis Encephalitis and West Nile Viruses in Free Ranging Birds during a Human Encephalitis Outbreak in Argentina.

St. Louis encephalitis virus (SLEV) (Flavivirus) is a reemerging arbovirus in the southern cone of South America. In 2005, an outbreak of SLEV in central Argentina resulted in 47 human cases with 9 deaths. In Argentina, the ecology of SLEV is poorly understood. Because certain birds are the primary amplifiers in North America, we hypothesized that birds amplify SLEV in Argentina as well. We compared avian SLEV seroprevalence in a variety of ecosystems in and around Córdoba city from 2004 (before the epidemic) and 2005 (during the epidemic). We also explored spatial patterns to better understand the local ecology of SLEV transmission. Because West Nile virus (WNV) was also detected in Argentina in 2005, all analyses were also conducted for WNV. A total of 980 birds were sampled for detection of SLEV and WNV neutralizing antibodies. SLEV seroprevalence in birds increased 11-fold from 2004 to 2005. Our study demonstrated that a high proportion (99.3%) of local birds were susceptible to SLEV infection immediately prior to the 2005 outbreak, indicating that the vertebrate host population was primed to amplify SLEV. SLEV was found distributed in a variety of environments throughout the city of Córdoba. However, the force of viral transmission varied among sites. Fine scale differences in populations of vectors and vertebrate hosts would explain this variation. In summary, we showed that in 2005, both SLEV and to a lesser extent WNV circulated in the avian population. Eared Dove, Picui Ground-Dove and Great Kiskadee are strong candidates to amplify SLEV because of their exposure to the pathogen at the population level, and their widespread abundance. For the same reasons, Rufous Hornero may be an important maintenance host for WNV in central Argentina. Competence studies and vector feeding studies are needed to confirm these relationships.

Neutralising antibodies for Mayaro virus in Pantanal, Brazil.

The Pantanal hosts diverse wildlife species and therefore is a hotspot for arbovirus studies in South America. A serosurvey for Mayaro virus (MAYV), eastern (EEEV), western (WEEV) and Venezuelan (VEEV) equine encephalitis viruses was conducted with 237 sheep, 87 free-ranging caimans and 748 equids, including 37 collected from a ranch where a neurologic disorder outbreak had been recently reported. Sera were tested for specific viral antibodies using plaque-reduction neutralisation test. From a total of 748 equids, of which 264 were immunised with vaccine composed of EEEV and WEEV and 484 had no history of immunisation, 10 (1.3%) were seropositive for MAYV and two (0.3%) for VEEV using criteria of a ≥ 4-fold antibody titre difference. Among the 484 equids without history of immunisation, 48 (9.9%) were seropositive for EEEV and four (0.8%) for WEEV using the same criteria. Among the sheep, five were sero- positive for equine encephalitis alphaviruses, with one (0.4%) for EEEV, one (0.4%) for WEEV and three (1.3%) for VEEV. Regarding free-ranging caimans, one (1.1%) and three (3.4%), respectively, had low titres for neutralising antibodies to VEEV and undetermined alphaviruses. The neurological disorder outbreak could not be linked to the alphaviruses tested. Our findings represent strong evidence that MAYV and all equine encephalitis alphaviruses circulated in the Pantanal.

Neutralising antibodies for Mayaro virus in Pantanal, Brazil

The Pantanal hosts diverse wildlife species and therefore is a hotspot for arbovirus studies in South America. A serosurvey for Mayaro virus (MAYV), eastern (EEEV), western (WEEV) and Venezuelan (VEEV) equine encephalitis viruses was conducted with 237 sheep, 87 free-ranging caimans and 748 equids, including 37 collected from a ranch where a neurologic disorder outbreak had been recently reported. Sera were tested for specific viral antibodies using plaque-reduction neutralisation test. From a total of 748 equids, of which 264 were immunised with vaccine composed of EEEV and WEEV and 484 had no history of immunisation, 10 (1.3%) were seropositive for MAYV and two (0.3%) for VEEV using criteria of a ≥ 4-fold antibody titre difference. Among the 484 equids without history of immunisation, 48 (9.9%) were seropositive for EEEV and four (0.8%) for WEEV using the same criteria. Among the sheep, five were sero- positive for equine encephalitis alphaviruses, with one (0.4%) for EEEV, one (0.4%) for WEEV and three (1.3%) for VEEV. Regarding free-ranging caimans, one (1.1%) and three (3.4%), respectively, had low titres for neutralising antibodies to VEEV and undetermined alphaviruses. The neurological disorder outbreak could not be linked to the alphaviruses tested. Our findings represent strong evidence that MAYV and all equine encephalitis alphaviruses circulated in the Pantanal.

Reduced West Nile Virus Transmission Around Communal Roosts of Great-Tailed Grackle (Quiscalus mexicanus).

West Nile virus has caused several outbreaks among humans in the Phoenix metropolitan area (Arizona, southwest USA) within the last decade. Recent ecologic studies have implicated Culex quinquefasciatus and Culex tarsalis as the mosquito vectors and identified three abundant passerine birds-great-tailed grackle (Quiscalus mexicanus), house sparrow (Passer domesticus), and house finch (Haemorhous mexicanus)-as key amplifiers among vertebrates. Nocturnal congregations of certain species have been suggested as critical for late summer West Nile virus amplification. We evaluated the hypothesis that house sparrow (P. domesticus) and/or great-tailed grackle (Q. mexicanus) communal roost sites (n = 22 and n = 5, respectively) in a primarily suburban environment were spatially associated with West Nile virus transmission indices during the 2010 outbreak of human neurological disease in metropolitan Phoenix. Spatial associations between human case residences and communal roosts were non-significant for house sparrows, and were negative for great-tailed grackle. Several theories that explain these observations are discussed, including the possibility that grackle communal roosts are protective.

Nhumirim virus, a novel flavivirus isolated from mosquitoes from the Pantanal, Brazil.

We describe the isolation of a novel flavivirus, isolated from a pool of mosquitoes identified as Culex (Culex) chidesteri collected in 2010 in the Pantanal region of west-central Brazil. The virus is herein designated Nhumirim virus (NHUV) after the name of the ranch from which the mosquito pool was collected. Flavivirus RNA was detected by real-time RT-PCR of homogenized mosquitoes and from the corresponding C6/36 culture supernatant. Based on full-genome sequencing, the virus isolate was genetically distinct from but most closely related to Barkedji virus (BJV), a newly described flavivirus from Senegal. Phylogenetic analysis demonstrated that NHUV grouped with mosquito-borne flaviviruses forming a clade with BJV. This clade may be genetically intermediate between the Culex-borne flaviviruses amplified by birds and the insect-only flaviviruses.

Serological evidence of widespread circulation of West Nile virus and other flaviviruses in equines of the Pantanal, Brazil.

A recent study reported neutralizing antibodies to West Nile virus (WNV) in horses from four ranches of southern Pantanal. To extend that study, a serosurvey for WNV and 11 Brazilian flaviviruses was conducted with 760 equines, 238 sheep and 61 caimans from 17 local cattle ranches. Among the tested equines, 32 were collected from a ranch where a neurologic disorder outbreak had been recently reported. The sera were initially screened by using a blocking ELISA and then titrated by 90% plaque-reduction neutralization test (PRNT90) for 12 flaviviruses. Employing the criterion of 4-fold greater titer, 78 (10.3%) equines were seropositive for Ilheus virus, 59 (7.8%) for Saint Louis encephalitis virus, 24 (3.2%) for WNV, two (0.3%) for Cacipacore virus and one (0.1%) for Rocio virus. No serological evidence was found linking the neurological disease that affected local equines to WNV. All caimans and sheep were negative by blocking ELISA for flaviviruses. There were no seropositive equines for Bussuquara, Iguape, Yellow fever and all four Dengue virus serotypes. The detection of WNV-seropositive equines in ten ranches and ILHV and SLEV-seropositive equines in fourteen ranches of two different sub-regions of Pantanal is strong evidence of widespread circulation of these flaviviruses in the region.

Ilheus virus isolation in the Pantanal, west-central Brazil.

The wetlands of the Brazilian Pantanal host large concentrations of diverse wildlife species and hematophagous arthropods, conditions that favor the circulation of zoonotic arboviruses. A recent study from the Nhecolândia sub-region of Pantanal reported serological evidence of various flaviviruses, including West Nile virus and Ilheus virus (ILHV). According to the age of seropositive horses, at least three flaviviruses, including ILHV, circulated in the Brazilian Pantanal between 2005 and 2009. To extend this study, we collected 3,234 adult mosquitoes of 16 species during 2009 and 2010 in the same sub-region. Mosquito pool homogenates were assayed for infectious virus on C6/36 and Vero cell monolayers and also tested for flaviviral RNA by a group-specific real-time RT-PCR. One pool containing 50 non-engorged female specimens of Aedes scapularis tested positive for ILHV by culture and for ILHV RNA by real-time RT-PCR, indicating a minimum infection rate of 2.5 per 1000. Full-length genomic sequence exhibited 95% identity to the only full genome sequence available for ILHV. The present data confirm the circulation of ILHV in the Brazilian Pantanal.

Host selection of potential West Nile virus vectors in Puerto Barrios, Guatemala, 2007.

The selection of vertebrate hosts by Culex mosquitoes relative to West Nile virus (WNV) transmission in neotropical countries such as Guatemala is not described. This study determined the feeding patterns of Cx. quinquefasciatus and Cx. nigripalpus and estimated the relative contribution of two common and frequently infected wild bird species, Turdus grayi and Quiscalus mexicanus, to WNV transmission. Engorged mosquitoes were collected from rural and urban habitats after the dry and wet seasons in the Department of Izabal in 2007. Host selection by Cx. nigripalpus varied significantly between urban and rural habitats. Both Cx. quinquefasciatus and Cx. nigripalpus fed predominantly on chickens and other domestic animals. Blood meals from wild birds were rare, accounting for 1.1% of blood meals identified from Cx. quinquefasciatus and 6.5% of blood meals from Cx. nigripalpus. Transmission of WNV by these two mosquito species may be dampened by extensive feeding on reservoir-incompetent hosts.

West Nile virus ecology in a tropical ecosystem in Guatemala.

West Nile virus ecology has yet to be rigorously investigated in the Caribbean Basin. We identified a transmission focus in Puerto Barrios, Guatemala, and established systematic monitoring of avian abundance and infection, seroconversions in domestic poultry, and viral infections in mosquitoes. West Nile virus transmission was detected annually between May and October from 2005 to 2008. High temperature and low rainfall enhanced the probability of chicken seroconversions, which occurred in both urban and rural sites. West Nile virus was isolated from Culex quinquefasciatus and to a lesser extent, from Culex mollis/Culex inflictus, but not from the most abundant Culex mosquito, Culex nigripalpus. A calculation that combined avian abundance, seroprevalence, and vertebrate reservoir competence suggested that great-tailed grackle (Quiscalus mexicanus) is the major amplifying host in this ecosystem. West Nile virus transmission reached moderate levels in sentinel chickens during 2007, but less than that observed during outbreaks of human disease attributed to West Nile virus in the United States.

Serological detection of West Nile virus in horses and chicken from Pantanal, Brazil

In an effort to detect West Nile virus (WNV) in Brazil, we sampled serum from horses and chickens from the Pantanal region of the state of Mato Grosso and tested for flavivirus-reactive antibodies by blocking ELISA. The positive samples were further confirmed for serological evidence of WNV infection in three (8%) of the 38 horses and one (3.2%) of the 31 chickens using an 80% plaque-reduction neutralisation test (PRNT80). These results provide evidence of the circulation of WNV in chickens and horses in Pantanal.

Serological detection of West Nile virus in horses and chicken from Pantanal, Brazil.

In an effort to detect West Nile virus (WNV) in Brazil, we sampled serum from horses and chickens from the Pantanal region of the state of Mato Grosso and tested for flavivirus-reactive antibodies by blocking ELISA. The positive samples were further confirmed for serological evidence of WNV infection in three (8%) of the 38 horses and one (3.2%) of the 31 chickens using an 80% plaque-reduction neutralisation test (PRNT80). These results provide evidence of the circulation of WNV in chickens and horses in Pantanal.

Avian hosts of West Nile virus in Puerto Rico.

West Nile virus (WNV) ecology in neotropical ecosystems is poorly understood, and vertebrate hosts responsible for infecting mosquitoes remain unidentified throughout the Caribbean Basin. After a period of intense WNV transmission among sentinel chickens near Ceiba, Puerto Rico, we measured abundance of resident birds and species-specific prevalence of WNV infection. Taking the product of these measures indicates the relative number of WNV infections by species. Greater Antillean grackle (Quiscalus niger) accounted for the most WNV infections among birds in our 100-km(2) study site. In urban habitats, the house sparrow (Passer domesticus) was frequently infected. Immature birds less than one year of age were more likely to have detectable WNV-reactive antibodies than older birds of the same species.

West Nile virus infection of birds, Mexico.

West Nile virus (WNV) has caused disease in humans, equids, and birds at lower frequency in Mexico than in the United States. We hypothesized that the seemingly reduced virulence in Mexico was caused by attenuation of the Tabasco strain from southeastern Mexico, resulting in lower viremia than that caused by the Tecate strain from the more northern location of Baja California. During 2006-2008, we tested this hypothesis in candidate avian amplifying hosts: domestic chickens, rock pigeons, house sparrows, great-tailed grackles, and clay-colored thrushes. Only great-tailed grackles and house sparrows were competent amplifying hosts for both strains, and deaths occurred in each species. Tecate strain viremia levels were higher for thrushes. Both strains produced low-level viremia in pigeons and chickens. Our results suggest that certain avian hosts within Mexico are competent for efficient amplification of both northern and southern WNV strains and that both strains likely contribute to bird deaths.

Seroconversion for west Nile and St. Louis encephalitis viruses among sentinel horses in Colombia

We prospectively sampled flavivirus-naïve horses in northern Colombia to detect West Nile virus (WNV) and St. Louis encephalitis virus (SLEV) seroconversion events, which would indicate the current circulation of these viruses. Overall, 331 (34.1 percent) of the 971 horses screened were positive for past infection with flaviviruses upon initial sampling in July 2006. During the 12-month study from July 2006-June 2007, 33 WNV seroconversions and 14 SLEV seroconversions were detected, most of which occurred in the department of Bolivar. The seroconversion rates of horses in Bolivar for the period of March-June 2007 reached 12.4 percent for WNV and 6.7 percent for SLEV. These results comprise the first serologic evidence of SLEV circulation in Colombia. None of the horses sampled developed symptoms of encephalitis within three years of initial sampling. Using seroconversions in sentinel horses, we demonstrated an active circulation of WNV and SLEV in northern Colombia, particularly in the department of Bolivar. The absence of WNV-attributed equine or human disease in Colombia and elsewhere in the Caribbean Basin remains a topic of debate and speculation.