Louis Encephalitis Virus Research Articles

Epidemiology of Neuroinvasive Arboviral Disease in the United States, 1999–2007

From 1999-2007, the most common causes of neuroinvasive arboviral disease in the United States, after West Nile virus (WNV), were California (CAL) serogroup viruses, St. Louis encephalitis virus (SLEV), and eastern equine encephalitis virus (EEEV). The CAL serogroup virus disease was primarily reported from Appalachia and the upper Midwest, SLEV disease from southern states, and EEEV disease from areas along the Atlantic and Gulf coasts. Children accounted for 88% of CAL serogroup virus disease, whereas 75% of SLEV disease occurred among older adults. The EEEV disease had the highest case-fatality rate (42%). The incidence of CAL serogroup virus and EEEV disease remained stable before and after the detection of WNV in the United States in 1999. The SLEV disease declined 3-fold after 1999; however, SLEV disease has occurred in sporadic epidemics that make trends difficult to interpret. The CAL serogroup virus, SLEV, and EEEV disease are persistent public health concerns in the United States warranting ongoing prevention efforts.

Does Variation in <I>Culex</I> (Diptera: Culicidae) Vector Competence Enable Outbreaks of West Nile Virus in California?

Since the invasion of California by West Nile virus (family Flaviviridae, genus Flavivirus, WNV) in 2003, we have annually monitored vector competence for the NY99 strain in Culex tarsalis Coquillett, Culex pipiens quinquefasciatus Say, Culex p. pipiens L., and Culex stigmatosoma Dyar populations from four areas: deserts of Coachella Valley, densely urbanized maritime Los Angeles, southern San Joaquin Valley in Kern County, and southern Sacramento Valley near Davis in Sacramento County. Overall, Cx. stigmatosoma was the most competent vector species, followed by Cx. tarsalis and the Cx. pipiens complex. The median infectious dose (ID50) of WNV required to infect 50% of the F1 female progeny reared from wild-caught females, a measure of mesenteronal susceptibility, ranged between 5 and 8 log10 plaque forming units/ml and was not correlated with annual human case incidence or summer maximum likelihood mosquito infection estimates. Odds ratios comparing nonoutbreak years with referent outbreak years were variable and failed to show a distinct pattern for Cx. tarsalis or Cx. pipiens complex females. Apparently factors other than midgut susceptibility within the ranges we measured enabled WNV outbreaks in California. Culex populations remained competent for St. Louis encephalitis virus, indicating that the disappearance of this virus was not related to a loss of vector competence.

Does Variation in Culex (Diptera: Culicidae) Vector Competence Enable Outbreaks of West Nile Virus in California?

Since the invasion of California by West Nile virus (family Flaviviridae, genus Flavivirus, WNV) in 2003, we have annually monitored vector competence for the NY99 strain in Culex tarsalis Coquillett, Culex pipiens quinquefasciatus Say, Culex p. pipiens L., and Culex stigmatosoma Dyar populations from four areas: deserts of Coachella Valley, densely urbanized maritime Los Angeles, southern San Joaquin Valley in Kern County, and southern Sacramento Valley near Davis in Sacramento County. Overall, Cx. stigmatosoma was the most competent vector species, followed by Cx. tarsalis and the Cx. pipiens complex. The median infectious dose (ID50) of WNV required to infect 50% of the F1 female progeny reared from wild-caught females, a measure of mesenteronal susceptibility, ranged between 5 and 8 log10 plaque forming units/ml and was not correlated with annual human case incidence or summer maximum likelihood mosquito infection estimates. Odds ratios comparing nonoutbreak years with referent outbreak years were variable and failed to show a distinct pattern for Cx. tarsalis or Cx. pipiens complex females. Apparently factors other than midgut susceptibility within the ranges we measured enabled WNV outbreaks in California. Culex populations remained competent for St. Louis encephalitis virus, indicating that the disappearance of this virus was not related to a loss of vector competence.

A duplex real-time reverse transcriptase polymerase chain reaction assay for the detection of St. Louis encephalitis and eastern equine encephalitis viruses

A duplex TaqMan real-time reverse transcriptase polymerase chain reaction (PCR) assay was developed for the detection of St. Louis encephalitis virus (SLEV) and eastern equine encephalitis virus (EEEV), for use in human and vector surveillance. The respective targets selected for the assay were the conserved NS5 and E1 genes of the 2 viruses. Because of the insufficient number of NS5 sequences from SLEV strains in the GenBank database, we determined the sequence of an approximately 1-kb region for each of 25 strains of SLEV to select primers and probes in a conserved region. Our assay has a sensitivity of 5 gene copies (gc)/reaction for EEEV and 10 gc/reaction for SLEV, and its performance is linear for at least 6 log 10 gc. The assay is specific and detected all strains of SLEV (69) and EEEV (12) that were tested. An internal control ensures detection of efficient nucleic acid extraction and possible PCR inhibition.

Detection and Serotyping of Dengue Virus in Serum Samples by Multiplex Reverse Transcriptase PCR-Ligase Detection Reaction Assay

The detection and successful typing of dengue virus (DENV) from patients with suspected dengue fever is important both for the diagnosis of the disease and for the implementation of epidemiologic control measures. A technique for the multiplex detection and typing of DENV serotypes 1 to 4 (DENV-1 to DENV-4) from clinical samples by PCR-ligase detection reaction (LDR) has been developed. A serotype-specific PCR amplifies the regions of genes C and E simultaneously. The two amplicons are targeted in a multiplex LDR, and the resultant fluorescently labeled ligation products are detected on a universal array. The assay was optimized using 38 DENV strains and was evaluated with 350 archived acute-phase serum samples. The sensitivity of the assay was 98.7%, and its specificity was 98.4%, relative to the results of real-time PCR. The detection threshold was 0.017 PFU for DENV-1, 0.004 PFU for DENV-2, 0.8 PFU for DENV-3, and 0.7 PFU for DENV-4. The assay is specific; it does not cross-react with the other flaviviruses tested (West Nile virus, St. Louis encephalitis virus, Japanese encephalitis virus, Kunjin virus, Murray Valley virus, Powassan virus, and yellow fever virus). All but 1 of 26 genotypic variants of DENV serotypes in a global DENV panel from different geographic regions were successfully identified. The PCR-LDR assay is a rapid, sensitive, specific, and high-throughput technique for the simultaneous detection of all four serotypes of DENV.

Genetic variation of St. Louis encephalitis virus

St. Louis encephalitis virus (SLEV) has been regularly isolated throughout the Americas since 1933. Previous phylogenetic studies involving 62 isolates have defined seven major lineages (I–VII), further divided into 14 clades. In this study, 28 strains isolated in Texas in 1991 and 2001–2003, and three older, previously unsequenced strains from Jamaica and California were sequenced over the envelope protein gene. The inclusion of these new sequences, and others published since 2001, has allowed better delineation of the previously published SLEV lineages, in particular the clades of lineage II. Phylogenetic analysis of 106 isolates identified 13 clades. All 1991 and 2001–2003 isolates from Nueces, Jefferson and Harris Counties (Texas Gulf Coast) group in clade IIB with other isolates from these counties isolated during the 1980s and 1990s. This lack of evidence for introduction of novel strains into the Texas Gulf Coast over a long period of time is consistent with overwintering of SLEV in this region. Two El Paso isolates, both from 2002, group in clade VA with recent Californian isolates from 1998–2001 and some South American strains with a broad temporal range. Overall, these data are consistent with multiple introductions of SLEV from South America into North America, and provide support for the hypothesis that in most situations, SLEV circulates within a locality, with occasional incursions from other areas. Finally, SLEV has much lower nucleotide (10.1 %) and amino acid variation (2.8 %) than other members of the Japanese encephalitis virus complex (maximum variation 24.6 % nucleotide and 11.8 % amino acid).

Intensive Early Season Adulticide Applications Decrease Arbovirus Transmission Throughout the Coachella Valley, Riverside County, California

In the Coachella Valley of California the seasonal onset of St. Louis encephalitis virus (SLEV), western equine encephalomyelitis virus (WEEV), and West Nile virus (WNV) has been detected consistently at the shoreline of the Salton Sea near the community of North Shore. The timing and intensity of initial amplification in the Culex tarsalis Coquillett/wild bird cycle at this focus seemed closely linked to the subsequent dispersal of virus to the rest of the Coachella Valley and perhaps southern California. In 2004, an attempt was made to interrupt the amplification and dispersal of WNV using ground ultra-low volume (ULV) applications of Pyrenone 25-5. Although these localized treatments were started 1 month after the initial detection in April, surveillance indicated no dispersal from this focus at this time. However, these treatments appeared to have little effect, and WNV eventually was detected throughout the valley, with seven human cases reported in the urbanized upper valley near Palm Springs. In 2005, the initial detection of WNV at North Shore at the end of May was followed rapidly by dispersal throughout the valley precluding efforts at containment. Evaluation of ground and aerial applications at North Shore during May and June 2005, respectively, indicated variable kill of sentinel mosquitoes (overall mortality: ground, 43%; air, 34%) and limited control of the target Cx. tarsalis population. In 2006, aerial ULV applications with the same chemical were begun immediately following the first detection of virus in mid-April, resulting in an apparent reduction of Cx. tarsalis abundance and delay of WNV activity in the rural lower valley and a marked decline in transmission by Culex quinquefasciatus Say populations in the densely populated upper northwestern valley with no human cases reported.

45-Year-Old Woman With Recurrent Headache and Photophobia

45-Year-Old Woman With Recurrent Headache and Photophobia

Evolution and dispersal of St. Louis encephalitis virus in the Americas

Using a Bayesian coalescent approach on a dataset of 73 envelope gene sequences we estimated substitution rates and dates of divergence for St. Louis encephalitis virus (SLEV) in the Americas. We found significant rate heterogeneity among lineages, such that “relaxed” molecular clock models were much better supported than a strict molecular clock. The mean substitution rate estimated for all SLEV was 4.1 × 10 −4 substitutions/site/year (95% HPD 2.5–5.7)—higher than previous estimates that relied on the less well-suited strict clock. Mean substitution rates for individual lineages varied from 3.7 × 10 −4 to 7.2 × 10 −4 substitutions/site/year. For the first time we also assessed the magnitude and direction of viral gene flow within the Americas. The overall direction of gene flow during the period represented by the phylogeny is from South to North, and the region between 15°N and 30°N latitude appears to be the major source of virus for the rest of North America, which is consistent with migratory birds returning to their northern breeding grounds having acquired infection while wintering in the region of the Gulf of Mexico.

Characterization of mosquito-adapted West Nile virus

West Nile virus (WNV), a mosquito-borne flavivirus, has significantly expanded its geographical and host range since its 1999 introduction into North America. The underlying mechanisms of evolution of WNV and other arboviruses are still poorly understood. Studies evaluating virus adaptation and fitness in relevant in vivo systems are largely lacking. In order to evaluate the capacity for host-specific adaptation and the genetic correlates of adaptation in vivo, this study measured phenotypic and genotypic changes in WNV resulting from passage in Culex pipiens mosquitoes. An increase in replicative ability of WNV in C. pipiens was attained for the two lineages of WNV tested. This adaptation for replication in mosquitoes did not result in a replicative cost in chickens, but did decrease cell-to-cell spread of virus in vertebrate cell culture. Genetic analyses of one mosquito-adapted lineage revealed a total of nine consensus nucleotide substitutions with no accumulation of a significant mutant spectrum. These results differed significantly from previous in vitro studies. When St Louis encephalitis virus (SLEV), a closely related flavivirus, was passaged in C. pipiens, moderately attenuated growth in C. pipiens was observed for two lineages tested. These results suggest that significant differences in the capacity for mosquito adaptation may exist between WNV and SLEV, and demonstrate that further comparative studies in relevant in vivo systems will help elucidate the still largely unknown mechanisms of arboviral adaptation in ecologically relevant hosts.

Evaluation of St. Louis encephalitis virus/dengue virus type 4 antigenic chimeric viruses in mice and rhesus monkeys

To develop a live attenuated virus vaccine against St. Louis encephalitis (SLE) virus, two antigenic chimeric viruses were generated by replacing the membrane precursor and envelope protein genes of dengue virus type 4 (DEN4) with those from SLE with or without a 30 nucleotide deletion in the DEN4 3′ untranslated region of the chimeric genome. Chimeric viruses were compared with parental wild-type SLE for level of neurovirulence and neuroinvasiveness in mice and for safety, immunogenicity, and protective efficacy in rhesus monkeys. The resulting viruses, SLE/DEN4 and SLE/DEN4Δ30, had greatly reduced neuroinvasiveness in immunodeficient mice but retained neurovirulence in suckling mice. Chimerization of SLE with DEN4 resulted in only moderate restriction in replication in rhesus monkeys, whereas the presence of the Δ30 mutation led to over-attenuation. Introduction of previously described attenuating paired charge-to-alanine mutations in the DEN4 NS5 protein of SLE/DEN4 reduced neurovirulence in mice and replication in rhesus monkeys. Two modified SLE/DEN4 viruses, SLE/DEN4-436,437 clone 41 and SLE/DEN4-654,655 clone 46, have significantly reduced neurovirulence in mice and conferred protective immunity in monkeys against SLE challenge. These viruses may be considered for use as SLE vaccine candidates and for use as diagnostic reagents with reduced virulence.

Persistent West Nile virus transmission and the apparent displacement St. Louis encephalitis virus in southeastern California, 2003-2006.

West Nile virus (family Flaviviridae, genus Flavivirus, WNV) invaded the Colorado Desert biome of southern California during summer 2003 and seemed to displace previously endemic St. Louis encephalitis virus (family Flaviviridae, genus Flavivirus, SLEV, an antigenically similar Flavivirus in the Japanese encephalitis virus serocomplex). Western equine encephalomyelitis virus (family Togaviridae, genus Alphavirus, WEEV), an antigenically distinct Alphavirus, was detected during 2005 and 2006, indicating that conditions were suitable for encephalitis virus introduction and detection. Cross-protective "avian herd immunity" due to WNV infection possibly may have prevented SLEV reintroduction and/or amplification to detectable levels. During 2003-2006, WNV was consistently active at wetlands and agricultural habitats surrounding the Salton Sea where Culex tarsalis Coquillett served as the primary enzootic maintenance and amplification vector. Based on published laboratory infection studies and the current seroprevalence estimates, house sparrows, house finches, and several Ardeidae may have been important avian amplifying hosts in this region. Transmission efficiency may have been dampened by high infection rates in incompetent avian hosts, including Gamble's quail, mourning doves, common ground doves, and domestic pigeons. Early season WNV amplification and dispersal from North Shore in the southeastern portion of the Coachella Valley resulted in sporadic WNV incursions into the urbanized Upper Valley near Palm Springs, where Culex pipiens quinquefasciatus Say was the primary enzootic and bridge vector. Although relatively few human cases were detected during the 2003-2006 period, all were concentrated in the Upper Valley and were associated with high human population density and WNV infection in peridomestic populations of Cx. p. quinquefasciatus. Intensive early mosquito control during 2006 seemed to interrupt and delay transmission, perhaps setting the stage for the future reintroduction of SLEV.

Persistent West Nile Virus Transmission and the Apparent Displacement St. Louis Encephalitis Virus in Southeastern California, 2003-2006

West Nile virus (family Flaviviridae, genus Flavivirus, WNV) invaded the Colorado Desert biome of southern California during summer 2003 and seemed to displace previously endemic St. Louis encephalitis virus (family Flaviviridae, genus Flavivirus, SLEV, an antigenically similar Flavivirus in the Japanese encephalitis virus serocomplex). Western equine encephalomyelitis virus (family Togaviridae, genus Alphavirus, WEEV), an antigenically distinct Alphavirus, was detected during 2005 and 2006, indicating that conditions were suitable for encephalitis virus introduction and detection. Cross-protective "avian herd immunity" due to WNV infection possibly may have prevented SLEV reintroduction and/or amplification to detectable levels. During 2003–2006, WNV was consistently active at wetlands and agricultural habitats surrounding the Salton Sea where Culex tarsalis Coquillett served as the primary enzootic maintenance and amplification vector. Based on published laboratory infection studies and the current seroprevalence estimates, house sparrows, house finches, and several Ardeidae may have been important avian amplifying hosts in this region. Transmission efficiency may have been dampened by high infection rates in incompetent avian hosts, including Gamble’s quail, mourning doves, common ground doves, and domestic pigeons. Early season WNV amplification and dispersal from North Shore in the southeastern portion of the Coachella Valley resulted in sporadic WNV incursions into the urbanized Upper Valley near Palm Springs, where Culex pipiens quinquefasciatus Say was the primary enzootic and bridge vector. Although relatively few human cases were detected during the 2003–2006 period, all were concentrated in the Upper Valley and were associated with high human population density and WNV infection in peridomestic populations of Cx. p. quinquefasciatus. Intensive early mosquito control during 2006 seemed to interrupt and delay transmission, perhaps setting the stage for the future reintroduction of SLEV.

Arbovirus Transmission by Culex nigripalpus in Florida, 2005

Understanding the transmission patterns of West Nile and St. Louis encephalitis viruses (family Flaviviridae, genus Flavivirus, WNV and SLEV) could result in an increased ability to predict transmission risk to humans. To examine transmission patterns between vector and host, we trapped mosquitoes in three Florida counties from June to November 2005 by using chicken-baited lard can mosquito traps. These traps were used to monitor for presence of WNV and SLEV in mosquitoes and subsequent transmission of these viruses to chickens. In total, 166,615 female mosquitoes were sorted into 4,009 pools based on species and bloodfed status, and they were tested for presence of WNV and SLEV. Sera from 209 chickens were tested for WNV and SLEV antibodies. We detected eight WNV-positive Culex nigripalpus Theobald mosquito pools; SLEV was not detected in any pools. Six positive pools were collected in August and September from Duval County, one pool in September from Manatee County, and one pool in November from Indian River County. Of the eight chickens potentially exposed to WNV, antibodies were detected in only one chicken, indicating a low rate of transmission relative to the observed mosquito infection rates. Low virus transmission rates relative to infection rates would suggest that using sentinel chicken seroconversion data as a means of arbovirus surveillance may underestimate the prevalence of WNV in the mosquito population. However, using mosquito infection rates may overestimate the risk of arboviral transmission. A variety of factors might account for the observed low level of transmission including a lack of viral dissemination in mosquito vectors.

Arbovirus transmission by Culex nigripalpus in Florida, 2005.

Understanding the transmission patterns of West Nile and St. Louis encephalitis viruses (family Flaviviridae, genus Flavivirus, WNV and SLEV) could result in an increased ability to predict transmission risk to humans. To examine transmission patterns between vector and host, we trapped mosquitoes in three Florida counties from June to November 2005 by using chicken-baited lard can mosquito traps. These traps were used to monitor for presence of WNV and SLEV in mosquitoes and subsequent transmission of these viruses to chickens. In total, 166,615 female mosquitoes were sorted into 4,009 pools based on species and bloodfed status, and they were tested for presence of WNV and SLEV. Sera from 209 chickens were tested for WNV and SLEV antibodies. We detected eight WNV-positive Culex nigripalpus Theobald mosquito pools; SLEV was not detected in any pools. Six positive pools were collected in August and September from Duval County, one pool in September from Manatee County, and one pool in November from Indian River County. Of the eight chickens potentially exposed to WNV, antibodies were detected in only one chicken, indicating a low rate of transmission relative to the observed mosquito infection rates. Low virus transmission rates relative to infection rates would suggest that using sentinel chicken seroconversion data as a means of arbovirus surveillance may underestimate the prevalence of WNV in the mosquito population. However, using mosquito infection rates may overestimate the risk of arboviral transmission. A variety of factors might account for the observed low level of transmission including a lack of viral dissemination in mosquito vectors.

Improvement of the purification of Saint Louis encephalitis virus NS2B-NS3 recombinant protease expressed in Escherichia coli

The NS2B-NS3 serine protease of Saint Louis Encephalitis virus (SLEV), a potential target for antiviral drug design, has been over-expressed as a recombinant His-tag protein in Escherichia coli for future structural determination. The production process resulted in a soluble protease with co-purification of DnaK, a bacterial molecular chaperone already described in E. coli protein expression. Two approaches were tested to remove this specific contaminant. The fusion protein bound to the purification resin was washed with MgATP plus soluble denatured E. coli proteins before elution, but this method proved to be poorly efficient due to a substantial loss of the targeted recombinant protease. After the immobilized metal affinity chromatography step, the use of gel permeation chromatography with addition of arginine in the mobile phase led to effective separation of the native viral protease from the DnaK aggregates. By this way, SLEV ΔNS2B-NS3pro protease was purified as a functional protein with a purity greater than 90% suitable for crystallization attempts.

Using Hydrologic Conditions to Forecast the Risk of Focal and Epidemic Arboviral Transmission in Peninsular Florida

The accurate forecasting and tracking of arboviral transmission is becoming increasingly critical for the early recognition and management of arboviral epidemics. Meteorological factors, especially rainfall and temperature, drive arboviral epidemics, but monitoring rainfall and temperature alone is not predictive of increased levels of vector-borne disease transmission. In Florida, model simulations of water table depth (WTD) provide a measure of drought, and they have been shown to provide an accurate forecast of arboviral transmission. Here, we tracked WTD in two peninsular Florida regions where focal West Nile virus (family Flaviviridae, genus Flavivirus, WNV) transmission was reported during 2004 and 2005. We compared the resulting WTD profiles with historical WTD simulations for Indian River County (IRC), FL, where two peninsular Florida St. Louis encephalitis virus epidemics had their epicenters in 1977 and 1990. In both of the regions where focal WNV transmission was reported during 2004 and 2005, the local WTD profiles approached the 1977 and 1990 IRC WTD profiles; however, differences in the local temporal sequence of hydrologic conditions were observed. These differences seem in part to explain why the focal WNV transmission during 2004 and 2005 failed to reach epidemic levels in peninsular Florida. These findings suggest that hydrologic monitoring, specifically WTD, may help determine the geographic extent, timing, and intensity of WNV transmission. We speculate that a more precise sequence of drought and wetting, including a secondary summer drying and wetting cycle, as occurred in IRC during 1977 and 1990, may provide the optimal hydrologic conditions for the expansion of an arbovirus outbreak from focal to epidemic. This study documents that monitoring hydrologic conditions, along with vector, avian amplification host, and virus population data, increases our ability to track and predict significant levels of arboviral transmission.

Eared Dove (Zenaida Auriculata, Columbidae) as Host for St. Louis Encephalitis Virus (Flaviviridae, Flavivirus)

St. Louis encephalitis virus (SLEV) is an emerging Flavivirus in South American countries. Its ecology and biological transmission cycles are scarcely known. Eared doves (Zenaida auriculata) have frequently been found infected by SLEV, and therefore, could be suspected as SLEV hosts. Thirty post-hatch-year eared doves were subcutaneously inoculated with the genotype V SLEV 78V-6507 viral strain and subsequently bled. No deaths or clinical signs of illness were observed in the inoculated doves. The viremia titers ranged from 2 to 5.5 log(10) plaque-forming units (PFU)/mL during 1-7 days postinoculation (dpi), the highest being observed on the 4th dpi. Mosquitoes were collected using can traps baited with chicken and eared doves for comparison. A total of 2792 mosquitoes belonging to 5 species were collected. Ninety percent of the mosquitoes collected in eared dove-baited can traps were Culex quinquefasciatus. Statistical differences were not observed in either Cx. quinquefasciatus (Chi(2) = 0.86; df = 1; p = 0.354) or in Cx. interfor (Chi(2) = 0.63; df = 1; p = 0.426) mosquitoes collected in both chicken- and eared dove-baited can traps. Considering that eared doves were frequently found naturally infected by SLEV, that they developed viremia titers higher than the minimum infection threshold needed to infect Cx. quinquefasciatus, and that these mosquitoes also fed on eared doves, they could be considered competent hosts for SLEV.

West Nile Virus in Birds, Argentina

West Nile Virus in Birds, Argentina

Molecular characterization of two rocio flavivirus strains isolated during the encephalitis epidemic in são paulo state, brazil and the development of a one-step rt-pcr assay for diagnosis

Rocio virus (ROCV) was responsible for an explosive encephalitis epidemic in the 1970s affecting about 1,000 residents of 20 coastland counties in São Paulo State, Brazil. ROCV was first isolated in 1975 from the cerebellum of a fatal human case of encephalitis. Clinical manifestations of the illness are similar to those described for St. Louis encephalitis. ROCV shows intense antigenic cross-reactivity with Japanese encephalitis complex (JEC) viruses, particularly with Ilheus (ILHV), St. Louis encephalitis, Murray Valley and West Nile viruses. In this study, we report a specific RT-PCR assay for ROCV diagnosis and the molecular characterization of the SPAn37630 and SPH37623 strains. Partial nucleotide sequences of NS5 and E genes determined from both strains were used in phylogenetic analysis. The results indicated that these strains are closely related to JEC viruses, but forming a distinct subclade together with ILHV, in accordance with results recently reported by Medeiros et al. (2007).