A survey of flaviviruses in birds in green areas of São Paulo, Brazil

Abstract:

INTRODUCTION:

This study aimed to investigate Flavivirus infection in birds captured in green areas of São Paulo.

METHODS:

Oropharyngeal swabs, cloacal swabs, and blood samples from 170 birds captured in two green areas in São Paulo, Brazil were subjected to real time-polymerase chain reaction (RT-qPCR) analysis of Flavivirus specific NS5 gene fragment.

RESULTS:

All samples were negative for the presence of viral ribonucleic acid (RNA).

CONCLUSIONS:

Despite the negative results, Flavivirus surveillance must be performed regularly due to favorable ecological conditions for virus circulation and transmission among birds in these areas and their close proximity to humans.

Keywords:
Birds; Flavivirus; Green areas

The genus Flavivirus comprises of approximately 70 viruses that can cause severe human diseases, such as West Nile fever, tick-borne encephalitis, Zika, and dengue fever¹. These viruses are maintained in the environment by transmission between hematophagous arthropods (mosquitoes or ticks) and susceptible vertebrate hosts, such as humans and birds². Interactions among these species can take place in green areas due to high biological diversity in these areas. In large cities such as São Paulo, intense urbanization has devastating impact on green areas³. According to Almeida & Vasconcellos, small pockets of forests still exist in São Paulo as public parks and conservation units⁴. A fauna survey performed in 81 green areas of the city showed 700 different species of animals, 372 of which were birds⁵. Forty one taxonomic categories of mosquitoes were found in 35 municipal parks of the city, including Culex species, a crucial mosquito genus for Flavivirus transmission⁶.

Migratory wild birds are considered the main hosts for certain flaviviruses, such as the Saint Louis encephalitis virus (SLEV) and West Nile virus (WNV)⁷. It is believed that these birds, specifically the Passeriformes, can carry pathogens during their migration, increasing the risk of Flavivirus spread in their staging areas⁸. Many species of migratory birds visit São Paulo annually; three of which arrive in Brazil from the Northern hemisphere between August and September, and return to the USA and Canada between April and May: osprey (Pandion haliaetus), peregrine falcon (Falco peregrinus), and lesser yellowlegs (Tringa flavipes)⁵. Because of the interaction between migratory wild birds and local avifauna that favors the spread of Flavivirus and favorable conditions for mosquito infestation, the objective of this study was to investigate Flavivirus circulation in birds captured in green areas of São Paulo.

Between March 2012 and January 2013, mist nets were utilized to capture birds in two different green areas in São Paulo (Figure 1): Anhanguera Park, located in the Perus district (coordinates: UTM Córrego Alegre - 23 S X-317.106 Y-7.409.705), and Castanheiras Farm/Environmental Protection Area (APA) Bororé Colônia, located in the Grajaú district, Bororé peninsula (coordinates: UTM Córrego Alegre - 23 S X-331.691 Y-7.365.989). These nets were left open 30 hours per month, at dawns or evenings, over a 12-month period. Nets were set before sunrise (at 05:30) and checked every 30 min. Captured birds were placed inside a cloth bag to minimize stress. Biometry of the birds was evaluated and all birds received a metal ring with individual identification number. A total of 170 birds were captured: 88 at the Anhanguera Park and 82 at the Castanheiras Farm (Table 1). The majority of these birds belonged to the order Passeriformes (84.6%), followed by Piciformes (7.7%), Columbiformes (3.8%), and Apodiformes (3.7%). Among the species captured, only Vireo olivaceus is considered migratory; other species are resident and/or endemic of the areas.

Figure 1
Location of the study areas. Pink: Anhanguera park and Castanheiras Farm. Blue: other green areas of São Paulo. Modified from São Paulo⁵. The identification of the blue dots were removed from the picture.

Table 1
Species of birds captured from March 2012 to January 2013 at the Anhanguera Park and Castanheiras Farm.

Cloacal and oropharyngeal swabs were collected from captured birds and placed in cryotubes containing 300µL of viral transport media [0,2% bovine serum albumin (GIBCO^TM), 0,8% penicillin/streptomycin solution (Sigma-Aldrich, USA), 0,2% amphotericin B (Sigma-Aldrich, USA), and 10% glycerol (Sigma-Aldrich, USA) in phosphate buffered saline (PBS) pH 7.5]. After collection, the tubes were transferred to a liquid nitrogen tank (-196°C) for flash freezing the samples and subsequently stored at -80°C until processing. Birds recaptured in different months were subjected to another swab collection. Deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) were extracted using the NucliSENS(r) automated platform (EasyMAG(r); bioMérieux, France) according to the manufacturer's instructions. The extracted samples were reverse transcribed using the SuperScript VILO kit (Invitrogen, USA) according to the manufacturer's instruction.

The complementary deoxyribonucleic acid (cDNA) was subjected to real time-polymerase chain reaction (qPCR) to amplify a 200-bp fragment of the Flavivirus NS5 gene, according to a method described by Johnson et al.⁹. The PCR reactions were performed in a 25µL volume [2.5µL of cDNA, 12.5µL of Power SYBR Green PCR Master Mix (Applied Biosystems, USA), 1µL of each 10µM primer, and 8µL of UltraPure water (Invitrogen)]. The thermocycler parameters were as follows: 95°C for 15 min, 40 cycles of 94°C for 30 sec, and 60°C for 1 min, followed by a dissociation curve at the end. The melting curve analysis showed comparable T_m values to those verified by Moureau et al.¹⁰. In all analyses, RNA samples extracted from culture supernatant of dengue virus (DENV) types 2, 3, and 4 were included as positive controls and water was used as a negative control.

Complementary deoxyribonucleic acid from positive samples were subjected to a semi-nested Flavivirus generic PCR to amplify a 269-272-bp fragment from the NS5 gene. The first reaction was carried out in a final volume of 25µL (2.5µL of cDNA, 16.2µL of UltraPure water, 2.5µL of PCR Buffer, 1.5µL of 50mM MgCl₂, 0.5µL of 10mM dNTP mix, 0.75µL of each 10µM primer, and 0.3µL of 5U/µL Platinum Taq DNA Polymerase (Invitrogen)]. The thermocycler parameters were 95°C for 10 min; 40 cycles of 94°C for 30 sec, 50°C for 30 sec, and 72°C for 45 sec; and 72°C for 10 min. Subsequently, a 2.5-µL aliquot from the first reaction was used for the second PCR according to a method described by Cook et al.¹¹ in a final volume of 25µL (2.5µL of amplicon, 16.95µL of UltraPure water, 2.5µL of PCR Buffer, 0.75µl of 50mM MgCl₂, 0.5µL of 10mM dNTP mix, 0.75µL of each 10µM primer, and 0.3µL of 5U/µL Platinum Taq DNA Polymerase). The thermocycler parameters were: 94°C for 2 min; 30 cycles of 94°C for 30 sec, 50°C for 45 sec, and 72°C for 1 min; and 72°C for 10 min. The amplified product was subjected to electrophoresis in a 1.5% agarose gel. The gel was subsequently stained with ethidium bromide and visualized under ultraviolet (UV) light.

Positive samples were purified with the Invisorb Fragment CleanUp reagent (Stratec Biomedical, Germany) according to the manufacturer's protocol for agarose gel DNA fragment purification. The BigDye Terminator Ready Reaction Mix (Thermo Fisher Scientific, USA) was used for sequencing in a final volume of 20µL [8µL of the Big Dye Reaction Mix, 5µL of amplicon, 5.9µL of water, and 1.1µL of each primer (3.2µM) described by Cook et al.¹¹⁾ ]. The thermocycler parameters were: 96°C for 1 min; and 25 cycles of 96°C for 10 sec, 50°C for 5 min, and 60°C for 4 min. After precipitation using 75% isopropanol, samples were sequenced using the ABI PRISM^(r)3500 Genetic Analyzer automatic sequencer (Applied Biosystems). Sequences were analyzed using the BLAST tool (Basic Local Alignment and Search Tool; https://blast.ncbi.nlm.nih.gov/Blast.cgi) and BioEdit software (Ibis Biosciences, USA).

In total, 347 samples were collected: 163 oropharyngeal swabs, 165 cloacal swabs, and 19 blood samples. A sample was considered positive when the amplification curve reached over the defined threshold and had a melting temperature between 79.4 and 83.7°C. Samples were considered negative when a) no cycle threshold (Ct) value was obtained, or b) Ct value was obtained but the melting temperature was outside of the defined range. Among all analyzed samples, four were considered positive by RT-qPCR (Table 2).

Table 2
Birds positive for flavivirus by quantitative reverse transcription polymerase chain reaction.

Among the four samples, blood samples from two birds (ID 230 and 232) did not show the expected 250-bp band of the Flavivirus NS5 gene following a semi-nested PCR. Oropharyngeal samples from the other two birds (ID 87 and 211) displayed the expected band (data not shown), which suggested viral genome amplification. These samples were subjected to sequencing analysis. However, a Basic Local Alignment Search Tool (BLAST) analysis showed sequence similarity between nucleotides of the oropharyngeal samples of bird IDs 87 and 211 and the RNA pseudouridylate synthase (RPUSD2) gene of Ficedula albicollis, a migratory bird species in the order Passeriformes (data not shown). Both samples were obtained from one bird species, Pitangus sulphuratus, which also belongs to the order Passeriformes.

Despite favorable conditions for Flavivirus transmission, the lack of positive results in this study was expected because typically PCR can only detect virus in viremic animals¹². According to Komar et al^., the viremic period in birds lasts approximately seven days⁸. Even when flaviviruses are circulating, it was unlikely to capture viremic birds, especially when sample collection was only performed once. Semi-nested PCR was performed in this study to confirm positive results obtained by SYBR Green-based RT-qPCR. Despite the cost benefit of SYBR Green-based PCR, a significant number of false positive results may be obtained due to non-specific SYBR Green binding to dsDNA, resulting in non-specific amplicons and primer dimers. In the current study, the gene encoding for the RPUSD2 enzyme was amplified by SYBR Green-based PCR in samples from three different birds. A high degree of similarity between amplified sequences with only few mismatches can be explained by genomic differences among bird species. Based on results of the current study, primers used in this study may not be suitable for bird samples due to sequence similarity with endogenous DNA sequences of birds.

In 2015, among major arboviruses circulating in Brazil, four were flaviviruses (SLEV, DENV, WNV, and Zika virus), with Zika virus and DENV found in São Paulo¹³. In average, 16 birds were captured per month in each area during the study period, which may not be a representative of the local avifauna population. Many important species were not captured during the period, presumably due to the need for other trapping methods or unsuitable site and/or time to set up mist nets. All captured birds are considered resident and/or endemic. These birds may travel short distance in search for territory or food, but they do not cross countries and continents, reducing the chance of introducing new pathogens. However, the presence of other flaviviruses in endemic species was previously described, for example, Rocio virus (ROCV), which was detected in a Zonotrichia capensis specimen¹⁴. This species can be found in green areas of São Paulo, suggesting Flavivirus circulation among local avifauna species. Therefore, despite the negative results, our study could not rule out the presence of Flavivirus species circulating among birds in these areas. These findings supported those reported in another study, in which the presence of Flavivirus of medical importance was not detected in mosquitoes captured in several municipal parks in São Paulo¹⁵. Due to their potential for harboring Flavivirus species, it would be necessary to capture more birds for better Flavivirus surveillance. The surveillance should be performed frequently in these and other areas in São Paulo, Brazil.

Acknowledgments

We gratefully acknowledge the State of São Paulo Research Foundation [Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP), Programa de Pesquisas em Caracterização, Conservação, Restauração e Uso Sustentável da Biodiversidade (BIOTA Program), Grant 2010/51230-8) for providing financial support; Veterinary Medicine and Wild Animals Handling Technical Division [Divisão Técnica de Medicina Veterinária e Manejo da Fauna Silvestre (DEPAVE-3/SVMA/PMSP)] for capturing and identifying the birds; and Tropical Medicine Institute of São Paulo (IMT-SP), for sample processing.

• ¹
  Figueiredo MLG. Identificação de Flavivirus infectando culicídeos de 1999 a 2007 no Brasil. Tese de Doutorado em Ciências: Instituto de Ciências Biomédicas da Universidade de São Paulo, São Paulo; 2010. 2010; 60 p.
• ²
  Hubálek Z. An annotated checklist of pathogenic microorganisms associated with migratory birds. J Wildl Dis 2004; 40:639-659.
• ³
  Mello-Théry NA. Conservação de áreas naturais em São Paulo. Estud Av 2011; 25:175-188.
• ⁴
  Almeida AF, Vasconcelos MK (Coord). Fauna silvestre: quem são e onde vivem os animais na metrópole paulistana. Secretaria Municipal do Verde e do Meio Ambiente, São Paulo; 2007. 350 p.
• ⁵
  Diário Oficial da Cidade de São Paulo. Inventário da Fauna do Município de São Paulo. Secretaria Municipal do Verde e do Meio Ambiente, São Paulo. 2010; 55 (supl):1-114.
• ⁶
  Medeiros-Sousa AR, Ceretti-Júnior W, de Carvalho GC, Nardi MS, Araujo AB, Vendrami DP, et al. Diversity and abundance of mosquitoes (Diptera: Culicidae) in an urban park: larval habitats and temporal variation. Acta Trop 2015; 150:200-209.
• ⁷
  Peterson AT, Komar N, Komar O, Navarro-Siguenza A, Robbins MB, Martínez-Meyer E. West Nile virus in the New World: potential impacts on bird species. Bird Conserv Int 2004; 14:215-232.
• ⁸
  Komar N, Langevin S, Hinten S, Nemeth N, Edwards E, Hettler D, et al. Experimental infection of North American birds with the New York 1999 strain of West Nile virus. Emerg Infect Dis 2003; 9:311-322.
• ⁹
  Johnson N, Wakeley PR, Mansfield KL, McCracken F, Haxton B, Phipps LP, et al. Assessment of a novel real-time pan-flaviviruses RT-polymerase chain reaction. Vector Borne Zoonotic Dis 2010; 10:665-671.
• ¹⁰
  Moureau G, Temmam S, Gonzalez JP, Charrel RN, Grard D, de Lamballerie X. A real-time RT-PCR method for the universal detection and identification of Flaviviruses. Vector Borne Zoonotic Dis 2007; 7:467-477.
• ¹¹
  Cook S, Moureau G, Harbach RE, Mukwaya L, Goodger K, Ssenfuka F. Isolation of a novel species of flavivirus and a new strain of Culex flavivirus (Flaviviridae) from a natural mosquito population in Uganda. J Gen Virol 2009; 90:2669-2678.
• ¹²
  Banet-Noach C, Simanov L, Malkinson M. Direct (non-vector) transmission of West Nile Virus in geese. Avian Pathol 2003; 32:489-494.
• ¹³
  Figueiredo LTM. The recent arbovirus disease epidemic in Brazil. Rev Soc Bras Med Trop 2015; 48:233-234.
• ¹⁴
  de Souza Lopes O, de Abreu Sachetta L, Francy DB, Jakob WL, Calisher CH. Emergence of a new arbovirus disease in Brazil. III. Isolation of Rocio virus from Psorophora ferox (Humboldt, 1819). Am J Epidemiol 1981; 113:122-125.
• ¹⁵
  Fernandes LN, Paula MB, Araújo AB, Gonçalves EF, Romano CM, Natal D, et al. Detection of Culex flavivirus and Aedes flavivirus nucleotide sequences in mosquitoes from parks in the city of São Paulo, Brazil. Acta Trop 2016; 157:73-83.

• FAPESP, BIOTA Program, Grant 2010/51230-8

Publication Dates

• Publication in this collection
  Jul-Aug 2016

History

• Received
  27 Jan 2016
• Accepted
  26 May 2016