Louis Encephalitis Virus Research Articles

North American Culex pipiens and Culex quinquefasciatus are competent vectors for Usutu virus.

BackgroundUsutu virus (USUV) is a member of the Japanese encephalitis virus (JEV) serocomplex in the Flaviviridae family. Emergence of USUV in Europe has led to disease burdens in birds and created increasing concern for the potential zoonotic transmission to humans. Whilst USUV has not been detected in the New World, the identification of competent vector species in North America is critical in the assessment of the likelihood of its dispersal and establishment of enzootic transmission cycles. The objective of this study was to determine vector competence of potential mosquito vectors in North America for USUV. Three medically important mosquito species were selected for testing because of their involvement in the transmission of West Nile virus and St. Louis encephalitis virus, two related JEV serocomplex flaviviruses in the New World.Methodology/Principal findingsOral challenge of Culex pipiens, Culex quinquefasciatus, and Aedes albopictus was performed to determine the susceptibility and vector competence of North American mosquitoes for USUV. Infection status was monitored by the isolation of virus from homogenized mosquito tissues. The disseminated form of infection was demonstrated by the detection of infectious virus in the head, wings, and legs of infected mosquitoes. The presence of viral RNA in saliva of infected Cx. pipiens and Cx. quinquefasciatus indicated that both species are competent for transmission of USUV.Conclusions/SignificanceResults indicate that members of the Cx. pipiens complex are susceptible to USUV and competent for its transmission potential in North America in the event of its introduction. In contrast, Ae. albopictus were highly refractory to USUV infection, suggesting that this species is unlikely to contribute to USUV transmission in North America.

Production of recombinant NS1 protein and its possible use in encephalitic flavivirus differential diagnosis

Saint Louis encephalitis virus (SLEV) and West Nile virus (WNV) are two of the major causes of arboviral encephalitis in the Americas. The co-circulation of related flaviviruses in the Americas and prior vaccination against flaviviruses pose problems to the diagnostic specificity of serological assays due to the development of cross-reactive antibodies. An accurate diagnosis method capable of differentiating these related viruses is needed. NS1 is a glycosylated, nonstructural protein, of about 46 kDa which has a highly conserved structure. Anti-NS1 antibodies can be detected within 4–8 days after the initial exposure and NS1 is the least cross-reactive of the flaviviral antigens. This study was aimed to generate SLEV and WNV NS1 recombinants proteins for the development of a flavivirus diagnostic test. Local Argentinian isolates were used as the source of NS1 gene cloning, expression, and purification. The protein was expressed in Escherichia coli as inclusion bodies and further purified by metal-chelating affinity chromatography (IMAC) under denaturing conditions. Human sera from SLEV and WNV positive cases showed reactivity to the recombinant NS1 proteins by western blot. The unfolded NS1 proteins were also used as immunogens. The polyclonal antibodies elicited in immunized mice recognized the two recombinant proteins with differential reactivity.

Ilheus and Saint Louis encephalitis viruses elicit cross-protection against a lethal Rocio virus challenge in mice.

Rocio virus (ROCV) was the causative agent of an unprecedented outbreak of encephalitis during the 1970s in the Vale do Ribeira, Sao Paulo State, in the Southeast region of Brazil. Surprisingly, no further cases of ROCV infection were identified after this outbreak; however, serological surveys have suggested the circulation of ROCV among humans and animals in different regions of Brazil. Cross-protective immunity among flaviviruses is well documented; consequently, immunity induced by infections with other flaviviruses endemic to Brazil could potentially be responsible for the lack of ROCV infections. Herein, we evaluated the cross-protection mediated by other flaviviruses against ROCV infection using an experimental C57BL/6 mouse model. Cross-protection against ROCV infection was observed when animals had prior exposure to Ilheus virus or Saint Louis encephalitis virus, suggesting that cross-reactive anti-flavivirus antibodies may limit ROCV disease outbreaks.

Neuronal Degeneration in Mice Induced by an Epidemic Strain of Saint Louis Encephalitis Virus Isolated in Argentina.

Saint Louis encephalitis virus (SLEV) is a neglected flavivirus that causes severe neurological disorders. The epidemic strain of SLEV, CbaAr-4005, isolated during an outbreak in Córdoba city (Argentina), causes meningitis and encephalitis associated with neurological symptoms in a murine experimental model. Here, we identified the affected brain areas and the damage triggered by this neurotropic arbovirus. We performed a detailed analysis of brain neurodegeneration associated with CbaAr-4005 SLEV infection in mice. The motor cortex, corpus striatum and cerebellum were the most affected structures. Neurodegeneration was also found in the olfactory bulb, thalamus, hypothalamus, hippocampus, and hindbrain. SLEV infection triggered brain cell apoptosis as well as somatodendritic and terminal degeneration. In addition, we observed massive excitotoxic-like degeneration in many cortical structures. Apoptosis was also detected in the neuroblastoma cell line N2a cultured with SLEV. The results evidenced that SLEV CbaAr-4005 infection induced severe degenerative alterations within the central nervous system of infected mice, providing new information about the targets of this flavivirus infection.

Evaluation of an arboviral syndrome query used in Maricopa County, Arizona

ObjectiveTo evaluate Arizona’s arboviral syndromic surveillance protocol in Maricopa County.IntroductionTimely identification of arboviral disease is key to prevent transmission in the community, but traditional surveillance may take up to 14 days between specimen collection and health department notification. Arizona state and county health agencies began monitoring National Syndromic Surveillance Program BioSense 2.0 data for patients infected with West Nile virus (WNV), St. Louis encephalitis virus (SLEV), chikungunya, or dengue virus in August 2015. Zika virus was added in April 2016. Our novel methods were presented at the International Society for Disease Surveillance 2015 Annual Conference. [1] Twice per week, we queried patient records from 15 Maricopa County BioSense-enrolled emergency department and inpatient hospitals for chief complaint keywords and discharge diagnosis codes. Our “Case Investigation Decision Tree” helped us determine whether records had a high or low degree of evidence for arboviral disease and necessitated further investigation. This study evaluated how Arizona’s protocol for conducting syndromic surveillance compared to traditional arboviral surveillance in terms of accuracy and timeliness in Maricopa County from August 2015 through December 2016.MethodsWe followed guidelines from the Centers for Disease Control and Prevention (CDC) to evaluate two major attributes of the protocol: accuracy [measured as positive predictive value (PPV) and sensitivity] and timeliness. [2] Arizona’s Medical Electronic Disease Surveillance Intelligence System (MEDSIS) was considered the “gold standard” system and BioSense was the test system. PPV was calculated as the proportion of records identified by BioSense that were reported to MEDSIS, regardless of final case classification. Sensitivity was the proportion of confirmed or probable cases in MEDSIS identified by BioSense. Though not all MEDSIS cases were seen at BioSense-reporting facilities, the sensitivity demonstrates how each query contributed to arboviral surveillance overall. We assessed timeliness in two ways: (1) the difference between the date when keywords or diagnosis codes were first identified by BioSense and the date the same patient was first reported to MEDSIS; and (2) the difference between the date the BioSense record was first reviewed by the Maricopa County Department of Public Health (MCDPH) syndromic surveillance team and the date the same patient was first investigated through MEDSIS by the MCDPH disease investigators. We assessed whether timeliness was affected by the method in which a record was identified in BioSense (i.e., chief complaint keyword or discharge diagnosis code).ResultsThe arboviral syndromic surveillance queries identified 62 records during the evaluation period (Table). For each arboviral query, the proportion of BioSense records that were also reported through MEDSIS ranged from 25.0% to 32.4%, except chikungunya, which had a PPV of 0%. BioSense records that had a high degree of evidence for arboviral disease tended to have a higher PPV compared to those with low evidence. BioSense records that were not already reported to MEDSIS met neither clinical nor exposure criteria for the arboviral diseases and were not deemed a public health risk. The sensitivities of the WNV and Zika queries to detect confirmed or probable cases in MEDSIS were 8.2% and 5.6%, respectively, while SLEV, chikungunya, and dengue queries were 0%. On average, patients were reported to MEDSIS 7 days prior to BioSense identifying keywords or diagnosis codes. In addition, MEDSIS cases were investigated by MCDPH disease investigators 10 days prior to MCDPH syndromic surveillance team review of BioSense records, on average. The average time between MEDSIS report date and BioSense identification date was shorter for BioSense records identified by chief complaint keywords than by diagnosis codes (4 and 8 days after MEDSIS, respectively).ConclusionsArizona’s arboviral syndromic surveillance protocol provided MCDPH with situational awareness, but BioSense data were not available more quickly than traditional mandated reporting. Through this process, we reviewed patient records that mentioned arboviral diseases and confirmed that these reportable conditions were captured in our traditional surveillance system. The decision tree was effective at prioritizing records for further investigation. Timeliness may be improved by updating the queries to include more chief complaint keywords and reviewing BioSense more than twice per week. MCDPH plans to evaluate Arizona’s updated arboviral syndromic surveillance protocol, which was adapted for BioSense Platform’s Electronic Surveillance System for Early Notification of Community-based Epidemics (ESSENCE).

Evaluation of Argentinean Bird Species as Amplifying Hosts for St. Louis Encephalitis Virus (Flavivirus, Flaviviridae).

St.Louis encephalitis virus (SLEV) is an emerging human pathogen flavivirus in Argentina. Recently, it has reemerged in the United States. We evaluated the role as amplifying host of six resident bird species and analyzed their capacity as host during the 2005 encephalitis outbreak of SLEV in Córdoba. Eared Dove, Picui Ground Dove, and House Sparrow were the three species with highest host competence index. At a city level, Eared Dove and Picui Ground Dove were the most important amplifying hosts during the 2005 SLEV human outbreak in Córdoba city. This finding highlighted important differences in the SLEV ecology between Argentina and the United States. Characterizing and evaluating the SLEV hosts contribute to our knowledge about its ecology and could help us to understand the causes that promote its emergence as a human pathogen in South America.

Human and Equine Infection with Alphaviruses and Flaviviruses in Panamá during 2010: A Cross-Sectional Study of Household Contacts during an Encephalitis Outbreak.

Members of the genera Alphavirus (family Togaviridae) and Flavivirus (family Flaviridae) are important zoonotic human and equine etiologic agents of neurologic diseases in the New World. In 2010, an outbreak of Madariaga virus (MADV; formerly eastern equine encephalitis virus) and Venezuelan equine encephalitis virus (VEEV) infections was reported in eastern Panamá. We further characterized the epidemiology of the outbreak by studying household contacts of confirmed human cases and of equine cases with neurological disease signs. Serum samples were screened using a hemagglutination inhibition test, and human results were confirmed using plaque reduction neutralization tests. A generalized linear model was used to evaluate the human MADV and VEEV seroprevalence ratios by age (in tercile) and gender. Overall, antibody prevalence for human MADV infection was 19.4%, VEEV 33.3%, and Mayaro virus 1.4%. In comparison with individuals aged 2-20 years, people from older age groups (21-41 and > 41 years) were five times more likely to have antibodies against VEEV, whereas the MADV prevalence ratio was independent of age. The overall seroprevalence of MADV in equids was 26.3%, VEEV 29.4%, West Nile virus (WNV) 2.6%, and St. Louis encephalitis virus (SLEV) was 63.0%. Taken together, our results suggest that multiple arboviruses are circulating in human and equine populations in Panamá. Our findings of a lack of increase in the seroprevalence ratio with age support the hypothesis of recent MADV exposure to people living in the affected region.

Scented Sugar Baits Enhance Detection of St. Louis Encephalitis and West Nile Viruses in Mosquitoes in Suburban California.

Scented sugar baits deployed in California deserts detected early West Nile virus (WNV) transmission by mosquitoes, representing a potential improvement to conventional arbovirus surveillance that relies heavily on infection rates in mosquito pools. In this study, we expanded deployment of scented sugar baits into suburban Sacramento and Yolo (2015, 2016) and Riverside Counties (2016), California. The goal of the study was to determine whether scented sugar baits detect WNV and St. Louis encephalitis virus (SLEV) concurrent with mosquito infections in trapped pools in areas of high human density. Between 8 and 10% of sugar baits were WNV RNA positive in both study years across the three counties. In Riverside County, where SLEV re-emerged in 2015, 1% of sugar baits were SLEV positive in 2016. Rates of sugar bait positives were at least 100 times higher than infection rates in trapped mosquitoes in the same districts. The prevalence of sugar bait positives varied temporally and did not coincide with infections in mosquitoes collected at the same sites each week. WNV RNA positive sugar baits were detected up to 2 wk before and after concurrent surveillance detected infection in mosquito pools at the same sites. Sugar baits also detected WNV in Riverside County at locations where no WNV activity was detected in mosquito pools. Sugar baits generated between 0.8 and 1.2 WNV positives per $1,000 and can be more economical than carbon dioxide baited traps that produce 0.8 positives per $1,000. These results indicate that the sugar bait approach enhances conventional arbovirus surveillance in mosquitoes in suburban California.

Serological evidence of widespread exposure of Grenada fruit bats to chikungunya virus.

SummaryAntibody detection against selected potentially zoonotic vector‐borne alphaviruses and flaviviruses was conducted on sera from bats from all six parishes in Grenada, West Indies. Sera were tested for (i) antibodies to flaviviruses West Nile virus, St. Louis encephalitis virus, Ilhéus virus, Bussuquara virus (BSQV), Rio Bravo virus and all four serotypes of dengue virus (DENV) by plaque reduction neutralization test (PRNT); (ii) antibodies to alphaviruses western equine encephalitis virus, Venezuelan equine encephalitis virus and eastern equine encephalitis virus by epitope‐blocking enzyme‐linked immunosorbent assay (ELISA); and (iii) antibodies to the alphavirus chikungunya (CHIKV) by PRNT. Two species of fruit bats were sampled, Artibeus jamaicensis and Artibeus lituratus, all roosting in or within 1,000 m of human settlements. Fifteen (36%) of the 42 bats tested for neutralizing antibodies to CHIKV were positive. The CHIKV‐seropositive bats lived in localities spanning five of the six parishes. All 43 bats tested for epitope‐blocking ELISA antibody to the other alphaviruses were negative, except one positive for Venezuelan equine encephalitis virus. All 50 bats tested for neutralizing antibody to flaviviruses were negative, except one that had a BSQV PRNT 80 titre of 20. The CHIKV serology results indicate that bats living close to and within human settlements were exposed to CHIKV in multiple locations. Importantly, bats for this study were trapped a year after the introduction and peak of the human CHIKV epidemic in Grenada. Thus, our data indicate that bats were exposed to CHIKV possibly during a time of marked decline in human cases.

West Nile and St. Louis encephalitis viral genetic determinants of avian host competence.

West Nile virus (WNV) and St. Louis encephalitis (SLEV) virus are enzootically maintained in North America in cycles involving the same mosquito vectors and similar avian hosts. However, these viruses exhibit dissimilar viremia and virulence phenotypes in birds: WNV is associated with high magnitude viremias that can result in mortality in certain species such as American crows (AMCRs, Corvus brachyrhynchos) whereas SLEV infection yields lower viremias that have not been associated with avian mortality. Cross-neutralization of these viruses in avian sera has been proposed to explain the reduced circulation of SLEV since the introduction of WNV in North America; however, in 2015, both viruses were the etiologic agents of concurrent human encephalitis outbreaks in Arizona, indicating the need to re-evaluate host factors and cross-neutralization responses as factors potentially affecting viral co-circulation. Reciprocal chimeric WNV and SLEV viruses were constructed by interchanging the pre-membrane (prM)-envelope (E) genes, and viruses subsequently generated were utilized herein for the inoculation of three different avian species: house sparrows (HOSPs; Passer domesticus), house finches (Haemorhous mexicanus) and AMCRs. Cross-protective immunity between parental and chimeric viruses were also assessed in HOSPs. Results indicated that the prM-E genes did not modulate avian replication or virulence differences between WNV and SLEV in any of the three avian species. However, WNV-prME proteins did dictate cross-protective immunity between these antigenically heterologous viruses. Our data provides further evidence of the important role that the WNV / SLEV viral non-structural genetic elements play in viral replication, avian host competence and virulence.

Seroprevalence of selected flaviviruses in free-living and captive capuchin monkeys in the state of Pernambuco, Brazil.

Mosquito-borne diseases such as dengue, yellow fever and, more recently, Chikungunya virus (CHIKV) and Zika virus (ZIKV) have a great impact in the public health. In addition, the presence of such viruses might have an impact on wild animal conservation as well as their possible role as animal reservoir. Here, we performed a serological survey searching for antibodies against a panel of flaviviruses [ZIKV, Dengue virus (DENV), Yellow Fever virus (YFV), West Nile virus (WNV), Saint Louis Encephalitis virus (SLEV), Ilheus virus (ILHV) and Rocio virus (ROCV)] using plaque reduction neutralization test (PRNT90 ) in both free-ranging and captive capuchin monkeys (Sapajus flavius and Sapajus libidinosus). Captive and free-living monkeys were sampled between June 2015 and January 2016 in the state of Pernambuco, including in the border with State of Paraíba, the epicentre of the ZIKV epidemics in Brazil. We have found neutralizing antibodies for ZIKV, DENV-1, DENV-2, DENV-3, DENV-4, YFV, ILHV and SLEV in both S.flavius and S.libidinosus samples. No positives samples were found for ROCV and WNV. Our results suggest that these flaviviruses might be circulating in capuchin monkey in the studied region. The possible presence of these viruses represents a risk for public health, as well as for animal conservation, especially for S.flavius which is a critically endangered species, facing high risk of extinction.

Seroprevalence of Flavivirus in horses in Chaco, Argentina. Circulation during 2013-2014

Seroprevalence of Flavivirus in horses in Chaco province, Argentina. Circulation during 2013-2014. Griselda Ines Oria 1,2, Lorena Ivana Spinsanti3, Veronica Laura Pirota1,4, Maria Florencia Martinez3, Ornela Sofia Stechina1,5, Eduardo Etchepare 6,7, Marta Silvia Contigiani3 and Marina Stein 1,5. Flaviviruses are responsible for several emerging mosquito-borne diseases in Argentina and in the Americas. Some of these diseases are zoonosis and were detected in recent years: West Nile virus (WNV) and Saint Louis encephalitis virus (SLEV) among them. WNV originated in Africa was detected for the first time in America in 1999 and it was first isolated from diseased and dead horses in 2006 in Argentina. Neutralizing antibodies from wild birds, humans and horses have been also detected in later years. SLEV re-emerged as a human pathogen in Argentina with the first epidemic in 2005. During this outbreak, viral strains were isolated from Culex mosquitoes and several studies have detected neutralizing antibodies from birds, human and horses, in the last ten years.The aim of this work is to provide information on the activity of the Flavivirus SLEV and WNV, identifying the infection in horses in three localities of the province of Chaco. From May 2013 to May 2014, serological surveys from 68 asymptomatic horses were conducted. Antibodies against SLEV (25 %) and WNV (8.82%) were detected. This is the first report of WNV circulation in equines from Chaco province and circulation of both viruses during the study period was demonstrated.

Efficacy of sea fennel (Crithmum maritimum L., Apiaceae) essential oils against Culex quinquefasciatus Say and Spodoptera littoralis (Boisd.)

The effective management of insect pests and vectors still represents a major challenge in current entomology. Culex quinquefasciatus Say is a key vector of lymphatic filariasis, Rift Valley fever, West Nile, St. Louis encephalitis and Western equine encephalitis virus. Recently, the importance of the eco-friendly control of C. quinquefasciatus larval population using plant-borne biopesticides, including essential oils (EOs), has been stressed. Spodoptera littoralis (Boisd.) also known as tobacco cutworm, is one of the most destructive moth agricultural pests in tropic and sub-tropic regions. Despite the fact that sea fennel (Crithmum maritimum L., Apiaceae) is considered as a promising biosaline crop, its potential for commercial cultivation has not yet been exploited. Notably, it has been reported that in coastal marine environments insect pests stay away from sea fennel plants. This aromatic plant is endowed with secretory structures as ducts and vittae storing volatile compounds. In the present study, the chemical composition of sea fennel essential oils from three different EO accessions, namely France (Brittany), Central and South Italy (Marche and Sicily regions) have been analysed by gas chromatography-mass spectrometry (GC–MS), and their toxicity against larvae of Cx. quinquefasciatus and S. littoralis was evaluated. Selected pure compounds from the essential oils mentioned above were also tested. The essential oils showed notable variability in chemical composition, being dominated by dillapiole and γ-terpinene (French EO), limonene and γ-terpinene (central Italy EO) and thymol methyl ether and γ-terpinene (Sicilian EO). As found out, the significantly most efficient essential oils were obtained from the seeds and aerial parts of plants of the French region. For these EOs, LD50 values were estimated as 62.3 and 71.7μg/larva for S. littoralis, respectively, and LC50 as 13.7 and 15.6μl/L for C. quinquefasciatus larvae, respectively. γ-Terpinene and myristicin were the most effective on C. quinquefasciatus larvae showing LC50 values <17μl/L. Overall, these findings shed light on the insecticidal potential of C. maritimum EO against mosquitoes and agricultural pests, allowing to identify the most effective chemotype, and to stimulate cultivation of this species as a source of botanical insecticides.

Wing variation in Culex nigripalpus (Diptera: Culicidae) in urban parks

BackgroundCulex nigripalpus has a wide geographical distribution and is found in North and South America. Females are considered primary vectors for several arboviruses, including Saint Louis encephalitis virus, Venezuelan equine encephalitis virus and Eastern equine encephalitis virus, as well as a potential vector of West Nile virus. In view of the epidemiological importance of this mosquito and its high abundance, this study sought to investigate wing variation in Cx. nigripalpus populations from urban parks in the city of São Paulo, Brazil.MethodsFemale mosquitoes were collected in seven urban parks in the city of São Paulo between 2011 and 2013. Eighteen landmark coordinates from the right wing of each female mosquito were digitized, and the dissimilarities between populations were assessed by canonical variate analysis and cross-validated reclassification and by constructing a Neighbor-Joining (NJ) tree based on Mahalanobis distances. The centroid size was calculated to determine mean wing size in each population.ResultsCanonical variate analysis based on fixed landmarks of the wing revealed a pattern of segregation between urban and sylvatic Cx. nigripalpus, a similar result to that revealed by the NJ tree topology, in which the population from Shangrilá Park segregated into a distinct branch separate from the other more urban populations.ConclusionEnvironmental heterogeneity may be affecting the wing shape variation of Cx. nigripalpus populations.

Host-Feeding Patterns of Culex stigmatosoma (Diptera: Culicidae) in Southern California.

Knowledge of the blood-feeding patterns exhibited by arthropod vectors is essential for understanding the complex dynamics of vector-borne disease transmission. Some species of mosquitoes belonging to the genus Culex have been implicated as having major roles in the transmission of arboviruses such as West Nile virus, Saint Louis encephalitis virus, and Western equine encephalitis virus. Although the host-feeding patterns for many of these Culex species are well studied, the host-feeding patterns of Culex stigmatosoma Dyar are relatively poorly studied, even though this species is suspected to be an important maintenance vector for West Nile virus and other arboviruses. In the current study, bloodmeals from 976 blood-engorged Cx. stigmatosoma, collected from 30 sites in southern California from 2009-2012, were processed for vertebrate host identification by nucleotide sequencing following polymerase chain reaction to amplify portions of the cytochrome oxidase I and cytochrome b genes of vertebrate animals. Vertebrate DNA was amplified, sequenced, and identified from a total of 647 Cx. stigmatosoma bloodmeals, revealing that 98.6% of bloodmeals were from birds, 1.2% from three mammal species, and a single bloodmeal was from a reptile species. In total, 40 different host species were identified. The greatest number of bloodmeals identified was from domestic chicken (Gallus gallus domesticus L.) (38% of bloodmeals), house sparrow (Passer domesticus L.) (23%), house finch (Haemorhous mexicanus Müller) (17%), American crow (Corvus brachyrhynchos L.) (4%), and mourning dove (Zenaida macroura L.) (3%). However, chicken bloodmeals were identified almost entirely from a single site where mosquito collection devices were placed in the near vicinity of confined domestic chickens. The strongly ornithophilic feeding behavior shown in this study for Cx. stigmatosoma supports the hypothesis that this mosquito species may be an important maintenance (or endemic) vector for arboviruses that circulate among susceptible birds.

Ensayos de sensibilidad de larvas de Aedes aegypti y Culex quinquefasciatus frente al nematodo Heterorhabditis bacteriophora en condiciones de laboratorio

Introduction: Aedes aegypti is the vector of dengue, yellow fever, Zika and Chikungunya viruses, and Culex quinquefasciatus is the vector of St. Louis and West Nile encephalitis viruses. Objective: To evaluate infectivity of Heterorhabditis bacteriophora N4 in C. quinquefasciatus and A. aegypti larvae under laboratory conditions. Materials and methods: Thirty second-instar larvae of the two mosquito species were exposed each to different doses (0:1, 1:1, 5:1, 15:1, 100:1, 500:1, 750:1 and 1,500:1) of nematode infective juveniles. Four replications per dose were performed. Results: Parasitism varied between 2.5 and 80 % in C. quinquefasciatus , and between 4.2 and 92.5 % in A. aegypti , with significant differences between doses (p<0.0001). DL50 were: 160.8 infective juveniles per larva for C. quinquefasciatus and 113.6 infective juveniles per larva for A. aegypti . In C. quinquefasciatus , 4 to 6 % of the infective juveniles developed to adults and in A. aegypti , 12- 61 %. In A. aegypti the emergence of new infective juveniles occurred with 100:1, 500:1, 750:1 and 1,500:1 infective juveniles per larva, and in C. quinquefasciatus , with 1,500:1 infective juveniles per larva. Melanization of infective juveniles was observed in both mosquito species. Conclusion: The susceptibility of these mosquito species to parasitism of an indigenous isolate of H. bacteriophora in the laboratory was demonstrated. Heterorhabditis bacteriophora N4 could be an efficient biological control agent.

Development of a model of Saint Louis encephalitis infection and disease in mice

BackgroundFlaviviruses are a genre of closely related viral pathogens which emerged in the last decades in Brazil and in the world. Saint (St.) Louis encephalitis virus (SLEV) is a neglected flavivirus that can cause a severe neurological disease that may lead to death or sequelae. St. Louis encephalitis pathogenesis is poorly understood, which hinders the development of specific treatment or vaccine.MethodsTo address this problem, we developed a model of SLEV infection in mice to study mechanisms involved in the pathogenesis of severe disease. The model consists in the intracranial inoculation of the SLEV strain BeH 355964, a strain isolated from a symptomatic human patient in Brazil, in adult immunocompetent mice.ResultsInoculated mice presented SLEV replication in the brain, accompanied by tissue damage, disease signs, and mortality approximately 7 days post infection. Infection was characterized by the production of proinflammatory cytokines and interferons and by leukocyte recruitment to the brain, composed mainly by neutrophils and lymphocytes. In vitro experiments indicated that SLEV is able to replicate in both neurons and glia and caused neuronal death and cytokine production, respectively.ConclusionsAltogether, intracranial SLEV infection leads to meningoencephalitis in mice, recapitulating several aspects of St. Louis encephalitis in humans. Our study indicates that the central nervous system (CNS) inflammation is a major component of SLEV-induced disease. This model may be useful to identify mechanisms of disease pathogenesis or resistance to SLEV infection.

Tissue tropism of Saint Louis encephalitis virus: Histopathology triggered by epidemic and non-epidemic strains isolated in Argentina

Saint Louis encephalitis virus (SLEV) reemerged in South America, and caused encephalitis outbreaks at the beginning of the 21st century. To enhance our knowledge about SLEV virulence, we performed comparative pathogenesis studies in Swiss albino mice inoculated with two different variants, the epidemic strain CbaAr-4005 and the non-epidemic strain CorAn-9275. Only the infection of mice with SLEV strain CbaAr-4005 resulted in high viremia, invasion of peripheral tissues including the lungs, kidney, and spleen, and viral neuroinvasion. This was associated with inflammatory pathology in the lungs, spleen, and brain as well as morbidity and mortality. In contrast, neither signs of desease nor viral replication were observed in mice infected with strain CorAn-9275. Interestingly, important loss of B cells and development of altered germinal centers (GC) were detected in the spleen of mice infected with strain CbaAr-4005, whereas mice infected with SLEV CorAn-9275 developed prominent GC with conserved follicular architecture, and neutralizing antibodies.

Efficacy of Deltagard®, Fyfanon®, and Evoluer™ 31-66 Against Culex quinquefasciatus In Harris County, Texas.

The comparative efficacy of ultra-low volume applications of deltamethrin, permethrin, and malathion against specific adult populations of Culex quinquefasciatus , a primary regional vector of Saint Louis encephalitis and West Nile virus, using field cage tests is described. At labeled application rates, the estimated mass median diameters for Fyfanon®, Evoluer™, and DeltaGard® were 18.32, 19.24, and 17.44 μm, respectively. No significant differences (P ≥ 0.01) in mean percent control were observed across all 3 wild populations and a susceptible Sebring strain tested with Fyfanon, DeltaGard, and Evoluer, providing 98.25%, 98.20%, and 97.24% mean percent control. Mean percent control for Sebring and 3 wild populations was 99.38%, 97.79%, 97.26%, and 97.15%, respectively, which were not significantly different from one another (P ≥ 0.01). Across all tests, highly significant differences (P ≤ 0.01) in mean percent control were obtained at 30.48, 60.96, and 91.44 m distances downwind, with mean percent control at 91.44 m being significantly less than that observed at 30.48 m, but not at 60.96 m. Individually, tests for DeltaGard, Fyfanon, and Evoluer resulted in no significant differences (P ≥ 0.01) in mean percent control between all mosquito populations tested, ranging from 96.47 to 100. However, Evoluer obtained significantly less (P ≤ 0.01) mean percent control at 91.44 m compared to DeltaGard and Fyfanon. Data suggest that the wild Cx. quinquefasciatus specimens collected were as susceptible as the laboratory Sebring strain. The use of Sebring as positive controls downwind strongly supported determination of the efficacy of all adulticides and susceptibility of wild populations tested.

Detection of the mosquito-borne flaviviruses, West Nile, Dengue, Saint Louis Encephalitis, Ilheus, Bussuquara, and Yellow Fever in free-ranging black howlers (Alouatta caraya) of Northeastern Argentina.

Several medically important mosquito-borne flaviviruses have been detected in Argentina in recent years: Dengue (DENV), St. Louis encephalitis (SLEV), West Nile (WNV) and Yellow Fever (YFV) viruses. Evidence of Bussuquara virus (BSQV) and Ilheus virus (ILHV) activity were found, but they have not been associated with human disease. Non-human primates can act as important hosts in the natural cycle of flaviviruses and serological studies can lead to improved understanding of virus circulation dynamics and host susceptibility. From July–August 2010, we conducted serological and molecular surveys in free–ranging black howlers (Alouatta caraya) captured in northeastern Argentina. We used 90% plaque-reduction neutralization tests (PRNT90) to analyze 108 serum samples for antibodies to WNV, SLEV, YFV, DENV (serotypes 1and 3), ILHV, and BSQV. Virus genome detection was performed using generic reverse transcription (RT)-nested PCR to identify flaviviruses in 51 antibody-negative animals. Seventy animals had antibodies for one or more flaviviruses for a total antibody prevalence of 64.8% (70/108). Monotypic (13/70, 19%) and heterotypic (27/70, 39%) patterns were differentiated. Specific neutralizing antibodies against WNV, SLEV, DENV-1, DENV-3, ILHV, and BSQV were found. Unexpectedly, the highest flavivirus antibody prevalence detected was to WNV with 9 (8.33%) monotypic responses. All samples tested by (RT)-nested PCR were negative for viral genome. This is the first detection of WNV-specific antibodies in black howlers from Argentina and the first report in free-ranging non-human primates from Latin-American countries. Given that no animals had specific neutralizing antibodies to YFV, our results suggest that the study population remains susceptible to YFV. Monitoring of these agents should be strengthened to detect the establishment of sylvatic cycles of flaviviruses in America and evaluate risks to wildlife and human health.