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.
Flaviviruses represent a group of mosquito-borne viruses in Brazil that are annually involved in a large number of human cases of dengue fever countrywide and sporadic local outbreaks of sylvatic yellow fever [1], [2]. Outbreaks caused by other flaviviruses have also been reported in the country. In the 1970s, the largest Brazilian epidemic of arbovirus encephalitis was caused by Rocio virus (ROCV) in southeast Brazil [3]. More recently, an outbreak of hemorrhagic manifestations was linked to Saint Louis encephalitis virus (SLEV) [4]. Sporadic human cases caused by other sylvatic flaviviruses, including Bussuquara virus (BSQV), Ilheus virus (ILHV) and Cacipacore virus (CPCV) have also been reported in Brazil [5], [6], [7]. Furthermore, yellow fever epizootics in howler monkeys were reported in 2008 and 2009. Approximately 200 carcasses tested positive for Yellow fever virus (YFV) and about 2000 deaths were reported [8]. Thirteen flaviviruses have been reported in Brazil, listed here in chronological order of discovery: YFV, ILHV, BSQV, SLEV, ROCV, CPCV, Dengue virus 1 (DENV-1) and Dengue virus 4 (DENV-4), Dengue virus 2 (DENV-2), Iguape virus (IGUV), Naranjal-like virus (NJLV), Dengue virus 3 (DENV-3) and Culex flavivirus (CXFV) [9], [10], [11], [12], [13], [14], [15], [16], [17], [18], [19], [20]. In 2009, serological evidence of West Nile virus (WNV) infection in Brazilian horses, was collected for the first time, in the Pantanal wetland region of Mato Grosso do Sul state (MS) [21]. The Pantanal wetland is a subtropical region of great biodiversity with strong potential for maintenance and evolution of mosquito-borne viruses. Comprising approximately 140,000 km2, the Pantanal is a vast sedimentary floodplain characterized by seasonal flooding which determines specific ecosystem processes, with the occurrence of plants and animals that are adapted to the annual shrinking and expansion of habitats due to the seasonal hydrological regime [22]. The region, which covers mainly Brazilian but also Paraguayan and Bolivian territories, is ecologically classified into 11 sub-regions according to vegetation, flooding and physiography. In Brazil, the Pantanal is located within the states of Mato Grosso (MT) and MS in the west-central region of the country [23]. Most of the flaviviruses isolated in Brazil are unknown or understudied in the Pantanal. A small number of investigations in the Nhecolândia Sub-region of the Pantanal, MS, have detected serological evidence for four flaviviruses, including ILHV, SLEV, WNV and CPCV [21], [24], [25]. Recently, three other serological studies reported the detection of neutralizing antibodies for WNV in equines from MT and again in MS, including from outside the boundaries of the Pantanal [26], [27], [28]. Complete surveys for flaviviruses in the Pantanal have not been reported. Therefore, we conducted a serosurvey for WNV and the 11 Brazilian flaviviruses of potential medical importance, utilizing equines, sheep and caimans as indicators.
The collections for this study were approved by the Animal Ethics Committee of Fundação Oswaldo Cruz (License CEUA-Fiocruz LW-1/12, protocol P-74/10-5) in compliance with the requirements of Brazilian Law 11794/2008, which rules on the scientific use of animals, including the principles of the Brazilian society of Science in laboratory animals. The collections were also approved by the Brazilian Institute of Environment and Natural Resources (licenses IBAMA 18363-1/2009 and 18363-2/2010). Caimans were captured in two ranches from sites where a high concentration of these animals was observed. Caiman blood samples were obtained by puncture of the internal jugular vein between the 1st and 2nd cervical vertebrae as described previously [29]. Information recorded about caimans included gender, weight and snout-vent length. Blood samples from equines, including horses, donkeys and mules, and from sheep were taken by puncture of the jugular vein in 14 and nine different ranches, respectively. Gender, age, tameness, breed, vaccination status, travel history outside of Pantanal and history of abnormalities, such as clinical signs involving the central or peripheral nervous system, were recorded for each equine sample. Information about gender, age, breed and travel history outside of Pantanal were also recorded for each sheep. All animals sampled appeared healthy except for one horse that presented with clinical signs suggestive of neurological disorder. Additionally, 36 horse samples collected in February 2010 from a ranch comprising roughly a 900-square-km area in the Nabileque Sub-region of Pantanal, also located in MS (Figure 1) were tested. An outbreak of an undiagnosed neurological syndrome had caused the death of 16 equines at this location from December 2009 to January 2010. All equines here were vaccinated for rabies and tested negative for Trypanosoma evansi, a common pathogen involved in incoordination and instability of hind limbs in Pantanal equines. These equines had not been vaccinated for equine encephalitis viruses at the time of the outbreak. After the first deaths, the herd was vaccinated for eastern and western equine encephalitis viruses. All samples were initially screened for flavivirus-reactive antibodies by blocking ELISA as described previously [30]. Briefly, the ability of the sera to block the binding of the flavivirus group-reactive monoclonal antibody 6B6C-1 to the cell lysate-derived antigen for WNV was compared to the blocking ability of negative control horse serum. Samples were considered seropositive when the inhibition values produced were ≥30%. Samples with inhibition values between 19 and 29% were repeated twice to confirm the negative results. Blocking ELISA-positive samples were then heat-inactivated and tested by PRNT90, as previously described [31], for WNV and 11 Brazilian flaviviruses. Briefly, serial two-fold dilutions that ranged from 1∶10–1∶320 or 1∶20–1∶640 of each blocking ELISA seropositive sample were tested for their ability to neutralize plaque formation by WNV, SLEV, ILHV, CPCV, BSQV, IGUV, YFV (vaccine strain 17D), ROCV, DENV-1, DENV-2, DENV-3 (recombinant ChimeriVax Dengue 3 virus) and DENV-4. Caiman and sheep samples that were negative by blocking ELISA were also tested by PRNT90 for WNV. All reference viruses used for PRNT were previously submitted to partial nucleotide sequencing of the N terminal region of NS5 gene to confirm viral identity. High identity scores were obtained with the following sequences deposited at GenBank: DENV-1 (FJ562106), DENV-2 (GQ398257), DENV-3 (recombinant ChimeriVax Dengue 3 virus) (JN811143), DENV-4 (GQ199880), ROCV (AY632542), SLEV (EF158048), ILHV (AY632539), YFV (JN628279), BSQV (AY632536), CPCV (AF013367), IGUV (AY632538), and WNV (JN819325). Serum was considered seropositive to a virus when it reduced at least 90% of the formation of plaques of this virus and its reciprocal neutralizing antibody titer was at least four-fold greater than what was observed for the other 11 tested flaviviruses. The PRNT is the most specific serological test for the differentiation of flavivirus infections in convalescent serum samples [32]. Type-specific antibodies against flaviviruses can be distinguished using the PRNT [33]. The conservative criterion of four-fold greater titer is based on the results of cross-neutralization tests with flaviviruses and their respective antisera used for studies of antigenic relationships [34], [35]. Two or more flaviviruses are distinct from each other by quantitative serological criteria (four-fold or greater differences between homologous and heterologous titers of both serum samples) [32]. The Cochran-Armitage Trend Test was used to test for positive trends in seroprevalence among age classes (StatXact 10. 0, Cytel Software Corporation, Cambridge, MA) All caiman and sheep serum samples were negative for flaviviruses-specific antibodies using the 6B6C-1 monoclonal antibody by blocking ELISA. When tested by PRNT90 for WNV, all caiman samples confirmed negative. Sixty caiman samples had PRNT90 titer <10 and one <40 for WNV (low sample volume of the latter required testing at a lowest dilution of 1∶40). From 238 sheep samples tested, 235 had PRNT90 titers <10, two showed low neutralizing antibodies titers of 20 and 10 and one <40. From 760 equine samples initially screened for flavivirus-reactive antibodies by blocking ELISA, 396 (52. 1%) were positive. When this subset was tested using PRNT90,332 (43. 7%) had neutralizing reactivity (PRNT90 titer ≥10) for ILHV, 251 (33%) for SLEV, 172 (22. 6%) for WNV, 139 (18. 3%) for CPCV, 130 (17. 1%) for ROCV, 62 (8. 2%) for IGUV, 14 (1. 8%) for YFV, 12 (1. 6%) for BSQV, four (0. 5%) for DENV-1, three (0. 4%) for DENV-2, one (0. 1%) for DENV-4 and none for DENV-3 (Table 1). Employing the criterion of 4-fold greater PRNT90 titer, 77 (10. 1%) equines were seropositive for ILHV, 59 (7. 8%) for SLEV, 24 (3. 2%) for WNV (see Table 2), two (0. 3%) for CPCV and one (0. 1%) for ROCV. From the remaining 232 blocking ELISA positive samples, 227 (29. 9%) were considered seropositive for undifferentiated flavivirus due to cross-reactivity and five (0. 6%) were considered seronegative with neutralizing antibodies titers <20 or <10 for all of the flaviviruses that were assayed by PRNT90. These five may represent infections by an unknown flavivirus. There were no seropositive equines using the four-fold antibody titer difference criterion for BSQV, YFV, IGUV, DENV-1, DENV-2, DENV-3 and DENV-4. Monotypic neutralization reactions (i. e. a sample that reacted with only one of the 12 flaviviruses in the panel) were detected for ILHV (n = 27), SLEV (n = 11) and WNV (n = 6). The only horse from Nhecolândia Sub-region that presented neurological signs was seropositive for undifferentiated flavivirus with the following PRNT90 titers: 80 for SLEV, 40 for ILHV, 20 for YFV, 10 for ROCV and <10 for all other flaviviruses tested. Neutralizing antibodies for WNV were detected in 172 equines from all 15 ranches that equines were tested. Considering the four-fold higher titer criterion as confirmation, the 24 confirmed WNV-seropositive equines were detected in ten ranches comprising an area of approximately 2,500 square-km in the Nhecolândia and Nabileque sub-regions of the Pantanal, located in MS (Figure 2). Besides the 24 WNV-seropositive equines, samples from 37 other equines presented PRNT90 titers for WNV ≥40 and among them 26 showed equal or higher titers for one of the other flaviviruses tested. For these 37 equines, the serology profile could not be interpreted to determine whether WNV reactivity was due to infection with WNV or cross-reactivity from other flavivirus infections. In fact, six of them were considered seropositive for one of the 11 other flavivirus tested (Table 3). Low level of neutralizing antibodies was detected for YFV, BSQV, IGUV, DENV-1, DENV-2 and DENV-4 even though none of the equines were considered infected with these viruses. In some cases, the PRNT90 titers for YFV, BSQV and IGUV exceeded 20. Both ILHV- and SLEV-seropositive equines were detected in 14 of the 15 ranches that equines were tested, comprising roughly a 3,100-square-km area. The only ranch that had no ILHV- and SLEV-seropositive equines was ranch PS (Figure 2). From 760 equines tested, 441 (58%) had date of birth available. Stratifying the serology results by age demonstrated that risk of exposure to flaviviruses increased with age for flaviviruses in general and for ILHV (1-tailed p<0. 001 for both), but not for SLEV (p = 0. 17) or WNV (p = 0. 46) (Figure 3). SLEV and WNV appeared to increase in prevalence with age for equines <9 years of age, but not for older animals. Equines seropositive for CPCV and ROCV were sampled in October 2009. The two CPCV-seropositive horses were five and seven years of age and were from ranch PQ, which comprises an area of approximately 140 square-km. The only horse that was seropositive for ROCV had no record of birth date and was from ranch PM that encompasses a 360-square-km area. At the end of 2009, an outbreak of neurologic disease among horses was reported from ranch PV located in the Nabileque Sub-region. From 36 equines sampled, 23 (63. 8%) were seropositive for flavivirus infection by blocking ELISA. When this subset was tested by PRNT90,11 (30. 5%) confirmed seropositive for SLEV, nine (25. 0%) for undifferentiated flavivirus (es), two (5. 5%) for WNV, and one (2. 8%) for ILHV (Table 4). Of the nine undifferentiated flavivirus infections, five had SLEV titers >20 and five had higher SLEV titers than other flaviviruses in the panel, indicating that a majority of these could have had a history of SLEV infection. In addition to the relatively low seroprevalence for WNV of just 5. 5% in this sample of 36 equines, four equines from this group that recovered from the recent neurologic syndrome were serologically negative for WNV (Table 4). In spite of recent vaccination against equine encephalitis viruses, most equines had low titers for equine encephalitis viruses. These results will be described separately.
In the present study, serological evidence of ILHV, SLEV, WNV, CPCV and ROCV circulation was detected in equines of the Pantanal. Severe disease has been linked to these viruses in humans [7], [13], [43], [58], [59] and for WNV also in equines [60]. WNV and SLEV circulation in the Pantanal appears to be a recent phenomenon. In the last three decades, the Pantanal has been impacted by the conversion of natural vegetation into agricultural fields and cattle pasture, with alteration and loss of natural habitats and biodiversity. Moreover, major negative impacts occur in uplands, with drastic deforestation of savanna vegetation, where main rivers feeding the Pantanal have their springs [22], [61]. These environmental changes in the Pantanal environs can directly impact the fluctuations of vector and vertebrate host populations, which could affect arbovirus transmission dynamics in the region. Loss of wetlands and other natural habitats typically leads to reduced biodiversity, and consequently increased risk of outbreaks of vector-borne diseases [62]. Preserving large wetland areas sustains biodiversity and may represent a valuable ecosystem-based approach for controlling WNV outbreaks [63]. Indeed, an outbreak of neurologic disease among equines reported herein occurred in a ranch that encompasses drier plateau ecosystem on the periphery of the Pantanal wetlands. Serology data suggest that an unknown flavivirus closely related to SLEV or a newly recognized equine-virulent strain of SLEV may be associated with this neurologic syndrome. Considering the highly conservative criteria used in the present study, the detection of seropositive equines for ILHV, SLEV, WNV, CPCV and ROCV is strong evidence for local circulation of these flaviviruses in the Pantanal of MS, Brazil. These flaviviruses should be included as differential diagnosis in future flavivirus serosurveys to be held in the region. For PRNT studies, not only these flaviviruses, but also YFV, BSQV and IGUV should be included. It appears that WNV circulated recently in the Pantanal. The detection of WNV-seropositive equines in at least ten ranches is strong evidence of recent widespread circulation, but not necessarily establishment of WNV in the region. Circulation of WNV in this area has not yet been confirmed by virus isolation. Because detection of antibodies is indirect evidence of flavivirus circulation and because novel flaviviruses may exist in the region, we encourage efforts to isolate viruses to confirm the circulation of these flaviviruses. In future investigations of equine epizootics, efforts should be concentrated on the collection of samples suitable for viral isolation.
West Nile virus is maintained in cycles between birds and mosquitoes and recently reemerged as a worldwide major public health and veterinarian concern as the cause of human and equine encephalitis outbreaks. Recent studies have reported serological evidence of West Nile virus circulation in Pantanal, west-central region of Brazil. However, considering the co-circulation of various cross-reactive flaviviruses in Brazil and that most of the flaviviruses isolated in Brazil are unknown or understudied in the Pantanal, serological results should be interpreted with caution. Therefore, we conducted a serosurvey for West Nile virus and the 11 Brazilian flaviviruses of potential medical importance, utilizing equines, sheep and caimans as indicators, including 32 equines collected from a ranch where a neurologic disorder had been recently reported among the equines. We found serological evidence of Ilheus, Saint Louis encephalitis, West Nile, Cacipacore and Rocio viruses in Pantanal equines. West Nile virus infection was not associated with the neurological disease of equines. 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.
emerging viral diseases
virology
biology
microbiology
Serological Evidence of Widespread Circulation of West Nile Virus and Other Flaviviruses in Equines of the Pantanal, Brazil