Serological evidence for Saint Louis encephalitis virus in free-ranging New World monkeys and horses within the upper Paraná River basin region, Southern Brazil

INTRODUCTION

Saint Louis encephalitis virus (SLEV) belongs to the genus Flavivirus, family Flaviviridae, which consists of approximately 70 virus species and subspecies distributed worldwide¹. Most flaviviruses are transmitted between susceptible vertebrates by hematophagous arthropods, in particular mosquitos^1,2. Flaviviruses are the most import causes of infectious diseases in humans from Brazil³; these viruses include Bussuquara, Cacipacore, dengue (serotypes 1, 2, 3, and 4), Iguape, Ilhéus, Rocio, Saint Louis encephalitis, and yellow fever^3,4.

SLEV may have initially originated in Central America⁵, but it has now disseminated throughout the Americas, with reports of its presence in the USA^6,7, Canada⁷, Argentina^8,9, Uruguay¹⁰, and Trinidad¹¹. In Brazil, SLEV was likely first isolated in the 1960s from a pool of Sabethes belisarioi mosquitoes captured on the Belém-Brasília Highway¹². Since then, this virus has been identified predominantly in humans^13–15 and horses^16–18 from Brazil.

Serological confirmation of SLEV in wildlife is very rare; it has been described in the white-tailed deer in the USA¹⁹, while seropositivity has been demonstrated in wild and sentinel animals and arthropods from both the Amazon region^20,21 and the State of São Paulo²², Brazil. Moreover, at least three epizootic incidents of SLEV have occurred in nonhuman primates, including sentinel Cebus monkeys in the Brazilian Amazon region²¹. Additionally, a serological survey conducted in French Guiana detected low levels of SLEV antibodies in free-ranging primates²³.

This study presents the findings of a serological investigation of SLEV in free-ranging New World monkeys and farm horses from southern Brazil.

METHODS

Study location

All monkeys used in this study were trap-captured within the Porto Rico County region, located between the northwestern region of the State of Paraná and the southeast region of the State of Mato Grosso do Sul, on the upper Paraná River in Brazil (Figure 1). This region consists of islands and sub-tropical forest reserves (where the animals were captured) that are protected by the Instituto Brasileiro do Meio Ambiente e dos Recursos Naturais Renováveis (IBAMA). The riparian forests of the islands of Mutum, Porto Rico, Gaivota, and Japonesa and the forests on the opposite shore of the Paraná River were included in this study. Porto Rico County is located in the northwestern region of the State of Paraná (22°46′20″S latitude and 53°16′01″W-GR longitude). This sub-tropical region has an average annual rainfall of 1,200-1,300mm and temperatures ranging from 16 to 29°C.

FIGURE 1 –  Google earth images illustrating the region where the research was conducted. The yellow pins indicate the coordinates where monkeys seropositive for Saint Louis encephalitis virus were trap-captured.  

RESULTS

Serological assay and virus isolation from nonhuman primates

According to the HI assay, SLEV antibodies were present in primates, specifically in 11.6% (5/43) of A. caraya, 12.5% (8/64) of S. nigritus, and 30.8% (8/26) of S. cay animals (Table 2). However, confirmation of SLEV infection by MNT was obtained for only 2.3% (1/43) of A. caraya, 6.3% (4/64) of S. nigritus and 15.4% (4/26) of S. cay primates (Table 2). Arboviruses were not isolated from any of the 133 primate-derived blood samples, although SLEV antibodies were detected by HI in 15.8% (21/133) of serum samples, with HI titers to SLEV ranging from 20 to 640 (Table 2 and Figure 2). Negative seroreactivity was observed for all other arboviruses.

TABLE 2 – Results of hemagglutination inhibition assays and mouse neutralization tests for free-ranging New World monkeys captured within the Porto Rico County region, Southern Brazil. 

HI result*(antibody titer)	Alouatta caraya		Sapajus nigritus		Sapajus cay		Total (male/female)
	male	female	male	female	male	female
Negative	21	17	40	16	10	8	112 (71/41)
20	—	2	2	1	2	2	9 (4/5)
40	2	1(2.7)	1	—	2(1.8/2.7)	—	6 (5/1)
80	—	—	1(1.9)	1(2.1)	—	1(2.7)	3 (1/2)
160	—	—	—	1(2.8)	—	—	1 (—/1)
320	—	—	—	—	—	—	—
640	—	—	1(3.5)	—	1(3.1)	—	2 (2/—)
Total	23	20	45	19	15	11	133 (83/50)

HI: hemagglutination inhibition.

^*HI test results: positive HI ≥ 20;

⁽ⁿ⁾neutralization logarithmic index (NLI): positive NLI ≥ 1.8.

FIGURE 2 –  Relationships between the total numbers of captured monkeys (by species) and positive results in the hemagglutination inhibition (HI) and mouse neutralization test (MNT) assays.  

The association of the results obtained based on NMT analysis of characteristics evaluated (species, sex, age, and the presence of horses within the same habitat) with the neutralization logarithm index (NLI) is summarized in Table 3. The prevalence of anti-SLEV antibodies was elevated in S. cay (15.4%; 4/26) relative to S. nigritus (6.3%; 4/64) and A. caraya (2.3%; 1/43) primates. Interestingly, negative seroreactivity of HI to SLEV antibodies was observed in infant and juvenile primates, while SLEV antibody serum prevalence was identified in sub-adult (13.3%; 2/15) and adult (9.2%; 7/76) animals, most likely due to their more extensive contact with this pathogen. Additionally, primates that live within proximity to farm horses demonstrated elevated seroreactivity to SLEV antibodies (15.4%; 4/26) compared to those living without potential contact with horses (4.8%; 5/107). However, no significant differences were observed based on species (ρ=0.1090), sex (ρ=0.7281), age (ρ=0.1876), and the presence of horses within the same habitat (ρ=0.0727).

TABLE 3 – Associations between the characteristics studied (species, sex, age, and the presence of horses within the same habitat) and the presence of anti-Saint Louis encephalitis virus antibodies (neutralization logarithm index) in serum samples of free-ranging New World Monkeys. 

Variables Species	Neutralization logarithmic index test						ORa	ρ-values
	positive		negative		total
	n	%	n	%	n	%
Alouatta caraya	1	2.32	42	97.68	43	32.33	Ac x Sn 0.36 (0.01-3.6)	0.63
Sapajus nigritus	4	6.25	60	93.75	64	48.12	Ac x Sc 0.13 (0.01-1.38)	0.12
Sapajus cay	4	15.38	22	84.61	26	19.55	Sn x Sc 0.37 (0.07-1.95)	0.38
Sex
male	5	6.02	78	93.98	83	62.41	0.74	0.72c
female	4	8.00	46	92.00	50	37.59	0.16 < OR < 3.48
Age
infant	—	—	7	100.00	7	5.27
juvenile	—	—	35	100.00	35	26.31	NC	0.18b
sub-adult	2	13.33	13	86.67	15	11.28
adult	7	9.21	69	90.79	76	57.14
Presence of horses in the same habitat
yes	4	15.38	22	84.62	26	19.55	3.71	0.07c
no	5	4.67	102	95.33	107	80.45	0.76 < OR < 17.79
Total (%)	9	6.77	124	93.23	133	100.00

Ac: Alouatta caraya; Sn: Sapajus nigritus; Sc: Sapajus cay; NC: not calculated; OR^a: odds ratio (inferior and superior limits);

^bchi-square;

^cFisher’s exact test.

DISCUSSION

The results of this study reveal that we have identified the presence of SLEV in several species of nonhuman primates and horses from southern Brazil. The classical HI and MNT methodologies used during this investigation are based on the conventional serologic diagnosis of flavivirus due to the presence of virus-specific antibodies in the serum. Similar serological strategies have been used to detect SLEV in primates²³, white-tailed deer^19,29, livestock¹¹, mules¹⁹, and horses^{8,10,11,17,18}. The importance of these results lies primarily in the observation of SLEV circulation in a completely new habitat within Brazil, considering that most cases of SLEV have been described within the Amazon region^4,20–22. However, SLEV was recently isolated from a horse in the State of Minas Gerais¹⁶, and SLEV has been serologically identified in both horses from Corumba, Central West Brazil¹⁷ and in patients from the State of São Paulo¹⁴. These results suggest a southern drift of SLEV from the Amazon region to other parts of continental Brazil, most likely attributable to migratory birds^16,21,22.

In the USA, SLEV causes encephalitis in approximately 100 human cases annually³⁰ and is one of the most common causes of arbovirus-induced disease³¹, with sporadic epidemics^6,30,31. However, this arboviral disease has different epidemiological features in Brazil, largely in terms of the relatively few cases of SLEV-induced encephalitis identified in humans^20,22. Additionally, most flaviviruses that occur in Brazil, with the exception of dengue virus, are predominantly maintained as sylvatic zoonotic diseases that occasionally produce infections in humans and domestic animals that have entered the ecosystems where these viruses occur⁴. Consequently, it can be speculated that this epidemiological difference might possibly be attributable to specific environmental and biological conditions within Brazil that alter the virulence or pathogenicity of SLEV in humans. Although the factors that are actively or otherwise associated with this phenomenon have not been fully elucidated, at least two theories should be considered. First, the elevated endemicity of other closely related flaviviruses that elicit cross-protection in humans, such as that occurring with dengue and yellow fever immunization, might easily result in the underdiagnosis of SLEV; this occurred in a study of 519 patients who were initially diagnosed as having dengue fever, but later molecular investigation confirmed the presence of SLEV in eight of them¹⁴. Secondly, the difficulty in efficiently recognizing and/or diagnosing SLEV encephalitis in patients at both public and private health services might also contribute to the reduced number of cases in Brazil, considering that patients may either be asymptomatic^4,16 or present with flu-like disease syndromes that can progress to acute or subacute meningeal and focal neurological manifestations³². Moreover, three patients from an outbreak of SLEV in northwestern São Paulo demonstrated hemorrhagic manifestations typical of dengue fever virus³³. Taken together, the confirmation of SLEV in several states in Brazil^13,14,16 in addition to the Amazon region may suggest dissemination due to migratory birds^16,22, as previously postulated. Furthermore, Culex declarator and Culex coronator are known vectors, while monkeys, sloths, armadillos, and marsupials are reservoirs of this virus in Brazil⁴. Therefore, the seropositivity of nonhuman primates and horses to SLEV demonstrated in this study in a region geographically distant from the Amazon region is of great concern, and additional investigation must be conducted to understand the dynamics associated with this virus in a new environment but in conventional hosts.

During this investigation, the majority (57.1%; 12/21) of seropositive monkeys demonstrated titers of SLEV according to HI that were ≥ 40, suggesting that these antibodies are attributable to the presence of SLEV and not cross-reactivity from contact with similar related viruses. Alternatively, in a seroepidemiological survey conducted in French Guiana, primates had low antibody titers to SLEV (HI <40) but elevated titers to yellow fever (HI >320), and the authors suggested that that SLEV antibodies identified could have been due to cross-reactions with yellow fever virus²³. Low titers of SLEV were also identified in horses, livestock, and wildlife from the island of Trinidad¹¹. However, during this investigation, seropositivity was only identified for SLEV, and negative results were obtained with all other viruses by HI, indicating that there was no cross-reaction with any similarly related virus.

In Argentina, SLEV studies have focused primarily on populations of mosquitoes^34,35, humans³⁶, and horses⁸, but the importance of monkeys in the sylvatic SLEV cycle has not been investigated. However, during this study, serological results confirmed the participation of nonhuman primates in the maintenance cycle of SLEV in southern Brazil. Consequently, these results most likely represent the first identification of SLEV in nonhuman primates beyond the Amazon region of Brazil.

Horses are commonly found in the region where sample collection was performed, largely on farms that are close to forested areas, and the relationships contributing to the presence of SLEV antibodies in these animals should be considered. Elevated prevalence rates of SLEV antibodies in horses from the Amazon¹⁸and Pantanal^17,18 regions have been described, but this report is the first description of SLEV in southern Brazil within an ecosystem that is very distant and quite different from the Amazon and Pantanal regions. Therefore, these results suggest that horses may participate as vertebrate hosts in the dissemination of SLEV and most likely should be considered amplifying sources of SLEV for primates, or vice versa. However, additional investigations must be performed to confirm this theory.

This report is the first describing the seroprevalence of specific SLEV antibodies in free-ranging monkeys within the State of Paraná, southern Brazil, and implicates nonhuman primates in the natural maintenance cycle of SLEV in the Southern Cone region, where an SLEV encephalitis outbreak was identified in northern Argentina³⁵. Future research will aim to identify local mosquito and human populations to establish their roles in the SLEV life cycle.