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Serological Evidence of Widespread Circulation of West Nile Virus and Other Flaviviruses in Equines of the Pantanal, Brazil  [PDF]
Alex Pauvolid-Corrêa ,Zilca Campos,Raquel Juliano,Jason Velez,Rita Maria Ribeiro Nogueira,Nicholas Komar
PLOS Neglected Tropical Diseases , 2014, DOI: 10.1371/journal.pntd.0002706
Abstract: 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.
Mutation of putative N-Linked Glycosylation Sites in Japanese encephalitis Virus Premembrane and Envelope proteins enhances humoral immunity in BALB/C mice after DNA vaccination
Yu Zhang, Puyan Chen, Ruibing Cao, Jinyan Gu
Virology Journal , 2011, DOI: 10.1186/1743-422x-8-138
Abstract: Japanese encephalitis virus (JEV) belongs to the genus Flavivirus, and the genus Flavivirus include many clinically important pathogens, such as dengue virus (DENV), West Nile virus (WNV), yellow fever virus, Murray Valley encephalitis virus, St.Louis encephalitis virus, and tick-borne encephalitis virus (TBEV). Japanese encephalitis virus (JEV) mostly causes infection of the central nervous system in humans and equines and stillbirths in swine [1,2]. The virus is zoonotic, cycling between birds and mosquitoes, and is transmitted to humans by infected mosquitoes. Since swine serve as a reservoir and amplifier of the virus [3], the development of a swine vaccine against JEV is a high priority, as it could help prevent epidemics in humans.JEV contains a single-stranded, plus-sense RNA genome of ~11 kb. It consists of a single open reading frame that codes for a large polyprotein of 3432 amino acids that is co- and post-translationally cleaved into three structural proteins (capsid, C; premembrane, prM; and envelope, E) and seven nonstructural proteins [4,5]. Envelope is the major structural protein, and makes up the surface of the avivirus particle. E protein has numerous neutralization epitopes, which mediate attachment to host cells, and a putative receptor-binding domain that induces the host immune response [6,7]. Though prM is able to fold independently of the E protein, correct folding of the E protein requires co-synthesis with prM [8]. PrM interacts with E to form prM-E heterodimers, which are important for the formation of immature virions[9,10], and the signal of the prM determine translocation and orientation of inserted protein, hence the topology of prM and E [11]. Therefore, the signal of the prM and the prM protein play an important role in maintaining its native conformation of E protein.N-linked glycans of viral proteins play important roles in modulating the immune response. Glycans can be important for maintaining the appropriate antigenic conformat
Isolation of Saint Louis Encephalitis Virus from a Horse with Neurological Disease in Brazil  [PDF]
Roberta Rosa,Erica Azevedo Costa,Rafael Elias Marques,Taismara Simas Oliveira,Ronaldo Furtini,Maria Rosa Quaresma Bomfim,Mauro Martins Teixeira,Tatiane Alves Paix?o,Renato Lima Santos
PLOS Neglected Tropical Diseases , 2013, DOI: 10.1371/journal.pntd.0002537
Abstract: St. Louis encephalitis virus (SLEV) is a causative agent of encephalitis in humans in the Western hemisphere. SLEV is a positive-sense RNA virus that belongs to the Flavivirus genus, which includes West Nile encephalitis virus, Japanese encephalitis virus, Dengue virus and other medically important viruses. Recently, we isolated a SLEV strain from the brain of a horse with neurological signs in the countryside of Minas Gerais, Brazil. The SLEV isolation was confirmed by reverse-transcription RT-PCR and sequencing of the E protein gene. Virus identity was also confirmed by indirect immunofluorescence using commercial antibodies against SLEV. To characterize this newly isolated strain in vivo, serial passages in newborn mice were performed and led to hemorrhagic manifestations associated with recruitment of inflammatory cells into the central nervous system of newborns. In summary this is the first isolation of SLEV from a horse with neurological signs in Brazil.
Serologic evidence of the recent circulation of Saint Louis encephalitis virus and high prevalence of equine encephalitis viruses in horses in the Nhecolandia sub-region in South Pantanal, Central-West Brazil
Pauvolid-Corrêa, Alex;Tavares, Fernando Neto;Costa, Eliane Veiga da;Burlandy, Fernanda Marcicano;Murta, Michele;Pellegrin, Aiesca Oliveira;Nogueira, Márcia Furlan;Silva, Edson Elias da;
Memórias do Instituto Oswaldo Cruz , 2010, DOI: 10.1590/S0074-02762010000600017
Abstract: as in humans, sub-clinical infection by arboviruses in domestic animals is common; however, its detection only occurs during epizootics and the silent circulation of some arboviruses may remain undetected. the objective of the present paper was to assess the current circulation of arboviruses in the nhecolandia sub-region of south pantanal, brazil. sera from a total of 135 horses, of which 75 were immunized with bivalent vaccine composed of inactive eastern equine encephalitis virus (eeev) and western equine encephalitis virus(weev) and 60 were unvaccinated, were submitted to thorough viral isolation, reverse transcriptase polymerase chain reaction (rt-pcr) and neutralization tests for saint louis encephalitis virus (slev), eeev, weev and mayaro virus (mayv). no virus was isolated and viral nucleic-acid detection by rt-pcr was also negative. nevertheless, the prevalence of neutralizing antibodies in horses older than seven months was 43.7% for slev in equines regardless of vaccine status, and 36.4% for weev and 47.7% for eeev in unvaccinated horses. there was no evidence of mayv infections. the serologic evidence of circulation of arboviruses responsible for equine and human encephalitis, without recent official reports of clinical infections in the area, suggests that the nhecolandia sub-region in south pantanal is an important area for detection of silent activity of arboviruses in brazil.
Experimental Passage of St. Louis Encephalitis Virus In Vivo in Mosquitoes and Chickens Reveals Evolutionarily Significant Virus Characteristics  [PDF]
Alexander T. Ciota,Yongqing Jia,Anne F. Payne,Greta Jerzak,Lauren J. Davis,David S.Young,Dylan Ehrbar,Laura D. Kramer
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0007876
Abstract: St. Louis encephalitis virus (SLEV; Flaviviridae, flavivirus) was the major cause of epidemic flaviviral encephalitis in the U.S. prior to the introduction of West Nile virus (WNV) in 1999. However, outbreaks of SLEV have been significantly more limited then WNV in terms of levels of activity and geographic dispersal. One possible explanation for these variable levels of activity is that differences in the potential for each virus to adapt to its host cycle exist. The need for arboviruses to replicate in disparate hosts is thought to result in constraints on both evolution and host-specific adaptation. If cycling is the cause of genetic stability observed in nature and arboviruses lack host specialization, then sequential passage should result in both the accumulation of mutations and specialized viruses better suited for replication in that host. Previous studies suggest that WNV and SLEV differ in capacity for both genetic change and host specialization, and in the costs each accrues from specializing. In an attempt to clarify how selective pressures contribute to epidemiological patterns of WNV and SLEV, we evaluated mutant spectra size, consensus genetic change, and phenotypic changes for SLEV in vivo following 20 sequential passages via inoculation in either Culex pipiens mosquitoes or chickens. Results demonstrate that the capacity for genetic change is large for SLEV and that the size of the mutant spectrum is host-dependent using our passage methodology. Despite this, a general lack of consensus change resulted from passage in either host, a result that contrasts with the idea that constraints on evolution in nature result from host cycling alone. Results also suggest that a high level of adaptation to both hosts already exists, despite host cycling. A strain significantly more infectious in chickens did emerge from one lineage of chicken passage, yet other lineages and all mosquito passage strains did not display measurable host-specific fitness gains. In addition, increased infectivity in chickens did not decrease infectivity in mosquitoes, which further contrasts the concept of fitness trade-offs for arboviruses.
An indirect immunofluorescence assay to detect antibodies against St. Louis Encephalitis virus
SPINSANTI, Lorena;RE, Viviana;AGUILAR, Javier;CONTIGIANI, Marta;
Revista do Instituto de Medicina Tropical de S?o Paulo , 2001, DOI: 10.1590/S0036-46652001000600008
Abstract: an in house indirect immmunofluorescence assay ( ifa ) in relation to neutralization (nt) reference test, was assessed as a fast and cheap method to carry out serological surveys for st. louis encephalitis virus (sle). sera obtained from 213 blood donors were analyzed by both tests. the prevalence of seropositivity obtained with ifa was lower than (30.98%) that observed on nt (41.78%). the relative specificity rate of ifa was 96.77% whereas its relative sensitivity rate was 69.66%. kappa index showed a good correlation between both tests. the results indicate that neutralization assay is still the serological test with the highest sensitivity and specificity relative rates for detecting antibodies against sle virus. nevertheless, the ifa could be useful as an alternative test in order to learn the circulation of the flavivirus genus in a certain area.
An indirect immunofluorescence assay to detect antibodies against St. Louis Encephalitis virus
SPINSANTI Lorena,RE Viviana,AGUILAR Javier,CONTIGIANI Marta
Revista do Instituto de Medicina Tropical de S?o Paulo , 2001,
Abstract: An in house indirect immmunofluorescence assay ( IFA ) in relation to neutralization (NT) reference test, was assessed as a fast and cheap method to carry out serological surveys for St. Louis Encephalitis virus (SLE). Sera obtained from 213 blood donors were analyzed by both tests. The prevalence of seropositivity obtained with IFA was lower than (30.98%) that observed on NT (41.78%). The relative specificity rate of IFA was 96.77% whereas its relative sensitivity rate was 69.66%. Kappa index showed a good correlation between both tests. The results indicate that neutralization assay is still the serological test with the highest sensitivity and specificity relative rates for detecting antibodies against SLE virus. Nevertheless, the IFA could be useful as an alternative test in order to learn the circulation of the Flavivirus genus in a certain area.
Silent Circulation of St. Louis Encephalitis Virus Prior to an Encephalitis Outbreak in Cordoba, Argentina (2005)  [PDF]
Luis Adrian Díaz ,Guillermo Albrieu Llinás,Ana Vázquez,Antonio Tenorio,Marta Silvia Contigiani
PLOS Neglected Tropical Diseases , 2012, DOI: 10.1371/journal.pntd.0001489
Abstract: St. Louis encephalitis virus is a complex zoonoses. In 2005, 47 laboratory-confirmed and probable clinical cases of SLEV infection were reported in Córdoba, Argentina. Although the causes of 2005 outbreak remain unknown, they might be related not only to virological factors, but also to ecological and environmental conditions. We hypothesized that one of the factors for SLE reemergence in Córdoba, Argentina, was the introduction of a new SLEV genotype (SLEV genotype III), with no previous activity in the area. In order to evaluate this hypothesis we carried out a molecular characterization of SLEV detections from mosquitoes collected between 2001 and 2004 in Córdoba city. A total of 315 mosquito pools (11,002 individuals) including 12 mosquitoes species were analyzed. Overall, 20 pools (8 mosquitoes species) were positive for SLEV. During this study, genotypes II, V and VII were detected. No mosquito pool infected with genotype III was detected before the 2005 outbreak. Genotype V was found every year and in the 8 sampled sites. Genotypes II and VII showed limited temporal and spatial activities. We cannot dismiss the association of genotype II and V as etiological agents during the outbreak. However, the silent circulation of other SLEV strains in Córdoba city before the 2005 outbreak suggests that the introduction of genotype III was an important factor associated to this event. Not mutually exclusive, other factors such as changes in avian hosts and mosquitoes vectors communities, driven by climatic and environmental modifications, should also be taken into consideration in further studies.
Comparison of Argentinean Saint Louis Encephalitis Virus Non-Epidemic and Epidemic Strain Infections in an Avian Model  [PDF]
Luis Adrián Diaz ,Nicole M. Nemeth,Richard A. Bowen,Walter R. Almiron,Marta S. Contigiani
PLOS Neglected Tropical Diseases , 2011, DOI: 10.1371/journal.pntd.0001177
Abstract: St. Louis encephalitis virus (SLEV, Flavivirus, Flaviviridae) is an emerging mosquito-borne pathogen in South America, with human SLEV encephalitis cases reported in Argentina and Brazil. Genotype III strains of SLEV were isolated from Culex quinquefasciatus mosquitoes in Cordoba, Argentina in 2005, during the largest SLEV outbreak ever reported in South America. The present study tested the hypothesis that the recent, epidemic SLEV strain exhibits greater virulence in birds as compared with a non-epidemic genotype III strain isolated from mosquitoes in Santa Fe Province 27 years earlier. The observed differences in infection parameters between adult House sparrows (Passer domesticus) that were needle-inoculated with either the epidemic or historic SLEV strain were not statistically significant. However, only the House sparrows that were infected with the epidemic strain achieved infectious-level viremia titers sufficient to infect Cx. spp. mosquitoes vectors. Furthermore, the vertebrate reservoir competence index values indicated an approximately 3-fold increase in amplification potential of House sparrows infected with the epidemic strain when pre-existing flavivirus-reactive antibodies were present, suggesting the possibility that antibody-dependent enhancement may increase the risk of avian-amplified transmission of SLEV in South America.
Genetic characterization of St. Louis encephalitis virus isolated from human in S?o Paulo, Brazil
Santos, Cecília Luiza Sim?es dos;Sallum, Maria Anice Mureb;Franco, Heitor Moreira;Oshiro, Fabíola Maiumi;Rocco, Iray Maria;
Memórias do Instituto Oswaldo Cruz , 2006, DOI: 10.1590/S0074-02762006000100011
Abstract: the molecular characterization of sph253157, a new strain of st. louis encephalitis virus (slev), isolated in 2004 from the first case of human infection recognized in the state of s?o paulo, brazil, is reported. the patient, presenting a febrile illness without neurological involvement, was hospitalized as a probable case of dengue fever. genomic rna was isolated from the supernatant of c6/36 cells infected with acute phase-serum specimen of the patient and the envelope gene was amplified by reverse-transcription-polymerase chain reaction. the complete nucleotide sequence of the envelope gene of this isolate was directly sequenced from the amplified products and compared with other brazilian and american slev strains. phylogenetic analyses were carried out under maximum likelihood criterion with outgroups both included and excluded. outgroups comprised four flavivirus of the japanese encephalitis group. phylogeny also included bayesian analysis. the results indicated that the new slev isolate belongs to lineage iii, being closely related to an argentinean strain recovered from culex sp. in 1979. it is concluded that there are at least 3 lineages of slev in brazil.
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