Serologic survey of West Nile virus in horses from Central-West, Northeast and Southeast Brazil.

Since the emergence of West Nile virus (WNV) in North America in 1999, there have been several reports of WNV activity in Central and South American countries. To detect WNV in Brazil, we performed a serological survey of horses from different regions of Brazil using recombinant peptides from domain III of WNV. Positive samples were validated with the neutralisation test. Our results showed that of 79 ELISA-positive horses, nine expressed WNV-specific neutralising antibodies. Eight of the infected horses were from the state of Mato Grosso do Sul and one was from the state of Paraíba. Our results provide additional evidence for the emergence of WNV in Brazil and for its circulation in multiple regions of the country.

West Nile virus (WNV), a member of the genus Flavivirus, is responsible for large outbreaks of disease in the Old World as well as in the New World (i.e., in North America) (Kilpatrick 2011). Since its emergence in New York City during an encephalitis outbreak in 1999, WNV has infected millions of humans and horses in North America (Petersen & Hayes 2008). Since its initial emergence, WNV activity has been reported in Central and South America, with specific antibodies detected in birds and horses (Komar & Clark 2006). In 2006, WNV was isolated from the brains of two dead horses in Argentina (Morales et al. 2006). In Brazil, WNV-specific neutralising antibodies were reported for the first time in horses from Nhecolândia, which is in the Pantanal region of the state of Mato Grosso do Sul (Pauvolid-Corrêa et al. 2011). Recently, serological evidence of WNV activity in horses and chickens has also been reported in the Brazilian Pantanal (Melandri et al. 2012). To date, the Brazilian health authorities have not reported any cases of WNV infection in humans or horses. Here, we report the results of a serological survey for WNV in different regions of Brazil.

Geographic locations of horse sera sample collection - Between 2004-2009, serum samples were collected from 753 horses from the states of São Paulo (SP) (n = 183), Minas Gerais (n = 15) and Rio de Janeiro (RJ) (n = 200) in Southeast Brazil, from the state of Paraíba (PB) (n = 88) in Northeast Brazil and from MS (n = 267) in Central-West Brazil. All participating horses were healthy at the time of blood collection and had no history of central nervous system infection or Fla-vivirus vaccination. Furthermore, the horses enrolled in this study had not travelled to areas in which WNV had been reported previously. A blood sample was collected from each animal by jugular venipuncture using vacutainers. Serum was separated by centrifugation and kept at -20ºC until use. The sample collection and handling procedures were approved by the Animal Ethical Committee of the University of São Paulo (USP).

Screening of horse sera by ELISA - Domain III of the flavivirus envelope protein is known to contain the critical antigenic epitopes that react with neutralising antibodies during Flavivirus infection. Therefore, we generated recombinant antigens consisting of domain III (rDIII) of WNV, Saint Louis encephalitis virus (SLEV) and Rocio virus (ROCV) in Escherichia coli (Chavez et al. 2010). An rDIII-ELISA was conducted according to a protocol previously standardised at the Virology Research Centre, School of Medicine of Ribeirão Preto, USP (Chavez et al. 2013). All sera were tested by rDIII ELISAs specific for SLEV, ROCV and WNV. Samples were considered positive at a dilution of 1:100 based on a cut-off point calculation.

Neutralisation test (NT) - Serum samples that tested positive in the WNV rDIII-ELISA, but not in the SLEV or ROCV ELISAs, were subsequently subjected to a NT, as previously described (Hawkes 1979). WNV strain NY-99 (kindly supplied by Dr Barbara W Johnson from the Centers for Disease Control and Prevention Fort Collins, CO, USA) was used in this assay. End-point titres were determined as the highest dilution of serum capable of neutralising 100% of a viral suspension at 100 TCID50.

Serum samples from 79 horses (10.5%) tested positive for WNV by rDIII-ELISA. Of these, only nine (11.3%) neutralised WNV, with reciprocal neutralising titres ranging from 16-128 in the NT (Table). Eight of these samples were isolated in MS and one was from PB (Figure). All other serum samples that tested positive in the WNV rDIII-ELISA, including those from animals of the southeastern states of SP (n = 19) and RJ (n = 34), tested negative in the NT.

TABLE

Positive samples to West Nile (WNV) virus in rDIII-WNV ELISA, their respective states and cities and neutralisation test (NT) titres

Species	Isolate	City	Hospital	qnrB-like allele	aac(6’)-Ib / aac(6’)-Ib-cr	ESBL genes	Phylogenetic group/clone	ST	MIC (µg/mL)
									NAL	CIP	LEV	GAT	GEN	TOB	AMK
Klebisella pneumoniae	CM4	CAB	H6	qnrB2	-/+	bla CTX-M-15	NC/Kp1	ST11	> 512	> 64	32	16	32	4	4
	CL4	CAB	H1	qnrB2	-/+	bla CTX-M-15	NC/Kp1	ST11	> 512	> 64	> 64	64	> 64	16	4
	I3	SF	H5	-	-/+	bla CTX-M-15	NC/Kp1	ST11	> 512	> 64	16	16	> 64	32	4
	I4	SF	H5	-	-/+	bla CTX-M-15	NC/Kp1	ST11	> 512	> 64	16	16	64	16	4
	CL6	CAB	H1	qnrB2	-/+	bla CTX-M-15	NC/Kp2	ST11	> 512	> 64	16	8	1	16	4
	CL9	CAB	H1	qnrB1	-/+	bla CTX-M-15	NC/Kp3	ST48	64	4	1	2	32	16	2
	T8	CH	H10	-	-/+	bla CTX-M-15	NC/Kp4	ST11	> 512	64	32	16	64	32	4
	CV1	CAB	H7	qnrB19	+/+	bla CTX-M-15/ bla CTX-M-2	NC/Kp5	ST11	> 512	> 64	64	32	> 64	64	32
	L5	CAB	H3	qnrB19	-/-	bla CTX-M-15	NC/ND	ST392	> 512	> 64	4	4	2	16	8
	B4	CAB	H4	qnrB2	-/-	bla CTX-M-15	NC/ND	ST11	> 512	64	> 64	64	0.5	1	1
	CV2	CAB	H7	-	-/+	bla CTX-M-2	NC/ND	ST15	> 512	64	8	4	< 0.5	4	4
	CM1	CAB	H6	qnrB19	-/+	bla CTX-M-2	NC/ND	ST11	> 512	> 64	64	32	> 64	> 64	> 256
	B5	CAB	H4	qnrB19	-/-	bla CTX-M-2	NC/ND	ST11	> 512	64	64	64	> 64	> 64	> 256
	CL7	CAB	H1	-	-/+	bla CTX-M-2	NC/ND	ST11	> 512	> 64	16	16	1	8	8
	M2	CAB	H7	qnrB19	-/-	bla CTX-M-2	NC/ND	ST11	> 512	> 64	> 64	64	2	16	> 256
	CL5	CAB	H1	-	-/+	bla CTX-M-8	NC/ND	ST14	64	8	2	4	1	16	8
Escherichia coli	L4	CAB	H3	-	-/+	bla CTX-M-15	B2/EC1a	ST131	> 512	> 64	16	16	1	16	8
	T1	CH	H10	-	-/+	bla CTX-M-15	B2/EC1a	ST131	> 512	> 64	64	16	> 64	> 64	16
	CM2	CAB	H6	-	-/+	bla CTX-M-15	B2/EC1b	ST131	> 512	> 64	8	8	> 64	32	8
	T3	CH	H10	-	-/+	bla CTX-M-15	B2/EC1b	ST131	> 512	64	32	8	1	1	2
	SM5	BA	H2	-	-/+	bla CTX-M-15	B2/EC1c	ST131	512	2	1	0.5	2	16	4
	M1	CAB	H7	-	-/+	bla CTX-M-15	A/EC2	ST410	> 512	> 64	32	16	> 64	64	32
	SM4	BA	H2	-	-/+	bla CTX-M-15	A/EC3	ST167	> 512	> 64	64	32	2	16	16
	T2	CH	H10	qnrB6	-/+	bla CTX-M-2	B1/EC4	ST297	> 512	> 64	> 64	> 64	2	32	4
	SM7	BA	H2	qnrB2	-/+	bla CTX-M-14	D/EC5	ST68	> 512	> 64	> 64	32	1	1	4
	SM8	BA	H2	qnrB2	-/-	bla CTX-M-14	D/EC5	ST68	> 512	> 64	64	32	< 0.5	0.5	0,5
Klebsiella oxytoca	C1	SF	H9	-	-/+	bla CTX-M-2	NC/ND	ND	128	2	4	2	8	16	2
	T4	CH	H10	qnrB10	-/-	bla CTX-M-2	NC/ND	ND	> 512	64	32	16	> 64	> 64	16
Proteus mirabilis	SM6	BA	H2	qnrB2	-/-	bla CTX-M-2	NC/ND	ND	> 512	> 64	> 64	64	32	8	4
	CX2	BA	H8	qnrB10	-/-	bla CTX-M-2	NC/ND	ND	128	2	4	2	8	16	32
	T12	CH	H10	qnrB19	-/-	bla CTX-M-2	NC/ND	ND	> 512	32	8	32	64	32	4
Providencia sp.	CL2	CAB	H1	qnrB2	-/-	bla CTX-M-2	NC/ND	ND	> 512	> 64	> 64	> 64	> 64	64	1
Enterobacter cloacae	CX1	BA	H8	qnrB10	-/-	bla PER-2/ bla SHV-12	NC/ND	ND	128	2	4	2	8	16	4

MS: state of Mato Grosso do Sul; PB: state of Paraíba.

Map of Brazil showing the locations where horses were sampled, including Pantanal region and cities, and the state of Paraíba (PB) where seropositive horses to West Nile virus were found. MG: state of Minas Gerais; MS: state of Mato Grosso; RJ: state of Rio de Janeiro; SP: state of São Paulo.

It is possible that many of the sera samples tested positive in ELISA as a result of assay cross-reactivity with other flaviviruses; these animals could have been infected by multiple flaviviruses during their lifetimes. In light of this, we considered only the serum samples that reacted in both the rDIII-WNV ELISA and WNV-NT as positives. Our results corroborate a report from 2011 showing that WNV-neutralising antibodies were found in 3% of the horses sampled in MS (Pauvolid-Corrêa et al. 2011). Recently, WNV-neutralising antibodies were found in horses and chickens sampled in the state of Mato Grosso (Melandri et al. 2012). It is possible that WNV was introduced, most likely by migratory birds, to the wildlife of the Pantanal region, which is a sanctuary for flora and fauna. It is well established that birds from North and South American countries migrate to certain Brazilian regions, including the Pantanal, each winter. Due to the large diversity of flora and fauna, as well as the high density of mosquitoes found in the Pantanal, this region may support the establishment and spread of WNV into other regions of the country (Pauvolid-Corrêa et al. 2011).

Between 2006-2008, serological studies in Central and South American countries reported WNV seroconversion in horses, humans and other vertebrates (Petersen & Hayes 2008). Interestingly, all of the WNV-positive horses in the present study were sampled in 2009, suggesting that they were likely infected prior to 2008. The spread of WNV between Central and South American countries likely contributed to the emergence of this virus in Brazil. It is possible that WNV has infected horses and/or humans in this region. However, to date, there have been no reports of human or horse diseases attributed to WNV in Brazil. Together, our results suggest that WNV is circulating and infecting horses in the Central-West Region of Brazil. The single positive sample from PB seems to be the first serological evidence of WNV circulation in Brazil outside of Pantanal and suggests that WNV is spreading toward the northeastern regions of the country. Health authorities should intensify surveillance for WNV in humans, horses and birds to evaluate the extent of the spread and to avoid the undetected emergence of WNV as a public health problem in Brazil.