Zika Virus Surveillance at the Human-Animal Interface in Brazil, Colombia, and Peru, 2017-2018

Abstract

Zika virus (ZIKV) was first discovered in 1947 in Uganda, but was not considered a public health threat until 2007 when found to be the source of epidemic activity in Asia. Epidemic activity spread to Brazil in 2014 and continues to spread throughout the tropical and subtropical regions of the Americas, where Aedes aegypti mosquitoes are abundant. Despite ZIKV being zoonotic in origin, information about potential vertebrate hosts and invertebrate vectors for ZIKV in the Americas and the role they play in virus maintenance and transmission is lacking. We conducted active surveillance of native primates, as well as other non-human vertebrates and mosquitoes for ZIKV in Brazil, Colombia, and Peru at established field sites up to 5 times per year, where there was evidence of recent or active ZIKV transmission in humans. Trapping efforts focused on abundant wildlife and domestic animals, as these would be candidate reservoirs or amplifiers of ZIKV. From February 2017 to March 2018, we collected 4,727 animal from 4 classes and over 47,000 mosquito samples from at least 13 different genera. We screened whole blood and pooled mosquito samples for ZIKV nucleic acid by a pan flavivirus real time RT-PCR and confirmed by ZIKV specific real time RT-PCR and/or sequencing. We tested plasma samples for presence of anti-ZIKV antibodies by plaque reduction neutralization testing (PRNT). Preliminary RT-PCR results reveal 8.3% (280/3,364) of animal and 36.8% (284/771) of pooled mosquito samples are positive for flavivirus; with one sample positive for ZIKV, a Howler monkey from Colombia. For preliminary serology, 8.9% (68/761) of animal plasma samples show evidence of anti-ZIKV antibodies by PRNT; 16.8% of birds and 8.6% of mammals tested showed evidence of previous ZIKV infection. Laboratory analysis for the remaining 55.8% (6,194/11,090) of individual and pooled samples is underway. Results of this study will facilitate a better understanding of ZIKV’s ability to establish a sylvatic cycle outside of human transmission, shape future surveillance strategies for ZIKV in tropical ecosystems, and inform the need for public health intervention efforts like vaccine development.

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Zika Virus Surveillance at the Human-Animal Interface in Brazil, Colombia, and Peru, 2017-2018
S.J. Salyer1 †, G.Porfirio2, R. Dezengrini-Slhessarenko3, T.O. Morgado4, V. Contreras5, S.Y. Rondon Robayo6, T.P. Quevedo7, E.Hannon8, J.R. Head1, H.M. Herrera2, G. Sabino-Santos9, L.M.S. Maia3, H. Contreras5, C.M. Leon6, S.A. Jenkins10, M.B. Parsons1, C.C. Netto11, H.G.
Dias12, E.H. Miranda13, M. Atencia5, M.J. Hoyos Loaiza6, C.N. Mores10, A. Balish1, S.M.B. Lima13, F.B. Santos12, G. Arrieta5, A. Link6,14, A.M. Bispo de Filippis12, L.T.M. Figueiredo9, A. Pauvolid-Corrêa12, S. Mattar5, C. González6, M.C. Guezala10, N. Komar8, J. Montgomery1
BACKGROUND
Despite Zika virus’s (ZIKV) zoonotic origins, information about potential vertebrate
hosts and invertebrate vectors for ZIKV in the Americas and the role they play in virus
maintenance and transmission is lacking. To help better understand the potential for
ZIKV to establish a sylvatic transmission cycle in the Americas, our team sought out to
identify:
Alternate vertebrate (i.e. non-human) hosts for ZIKV
Alternate mosquito vectors for ZIKV
Potential transmission networks through blood meal identification
1EISL Branch, Division of Global Health Protection, Center for Global Health, CDC-Atlanta; 2Universidade Católica Dom Bosco (UCDB), Campo Grande, Brazil; 3Faculdade de Medicina, Universidade Federal de Mato Grosso (UFMT), Cuiabá, Brazil; 4Hospital Veterinário, Universidade Federal de Mato Grosso (UFMT),
Cuiabá, Brazil; 5Instituto de Investigaciones Biológicas del Trópico-IIBT, Universidad de Córdoba, Monteria, Colombia; 6Centre for Research in Tropical Microbiology and Parasitology (CIMPAT), Department of Biological Sciences, Universidad de Los Andes, Bogotá, Colombia; 7Vysnova Partners Inc., Lima, Peru;
8Arbovirus Diseases Branch, Division of Vector-Borne Diseases, National Center for Emerging and Zoonotic Infectious Diseases, CDC-Ft. Collins, CO; 9Faculdade de Medicina, Universidade de São Paulo (USP), Ribeirão Preto, Brazil; 10U.S. Naval Medical Research Unit 6 (NAMRU-6) Lima, Peru; 11Centro de Reabilitação
de Animais Silvestres (CRAS), Campo Grande, Brazil; 12Instituto Oswaldo Cruz, Fundação Oswaldo Cruz (Fiocruz), Rio de Janeiro, Brazil; 13Bio-Manguinhos, Fundação Oswaldo Cruz (Fiocruz), Rio de Janeiro, Brazil; 14Departamento de Ciencias Biologicas y Facultad de Administracion, Universidad de Los Andes, Bogota,
Colombia; †Corresponding author: wig9@cdc.gov
RESULTS
From February 2017 to March 2018, each team completed
~ 4 campaigns, collecting samples from over 4,607 animals
from 200 different wild and domestic species, and 56,820
mosquito samples from over 100 species.
ACKNOWLEDGEMENTS
Division of Global Health Protection
Class No. Whole Blood
Samples
No. Screened by
Flavivirus rt RT-PCR
No.
Flavivirus rt RT-
PCR
Positive (%)
No. Tested by ZIKV
rt RT-PCR
No. ZIKV rt RT-
PCR Positive (%)
Positive for ZIKV
by sequencing
(%)
Amphibia 110 107 25 (23.4) 25 0 (0) 0 (0)
Mammalia 3075 2469 329 (13.3) 460 0 (0) 1 (0.3%)*
Aves 1070 1013 187 (18.5) 265 0 (0) 0 (0)
Reptilia 346 337 25 (7.4) 25 0 (0) 0 (0)
Total 4607 4368 567 (13.0) 818 0 (0) 0 (0)
Class Order2
No. Plasma
Samples
Collected
No.
Screened by
PRNT
No. ZIKV
Reactive (%)
No. DENV 3
Reactive (%)
No. ILHV
Reactive (%)
No. WNV
Reactive (%)
No. YFV
Reactive (%)
Amphibia
110 51 (20.0) 0 (0) 0 (0) 0 (0) 0 (0)
Anura 109 41 (25.0)
Aves 1069 459 23 (5.0) 0 (0) 0 (0) 0 (0) 0 (0)
Anseriformes 149 90 9 (10.0)
Cariamiformes 3 2 1 (50.0)
Galliformes 780 360 12 (3.3)
Rheiformes 6 6 1 (1.7)
Mammalia
3071 1303 64 (4.9) 1(1.6) 1(1.6) 2 (3.1) 0 (0)
Artiodactyla 704 226 22 (9.7)
Carnivora 838 480 13 (2.7) 1 (7.7)
Chiroptera 573 217 8 (3.7) 1 (12.5) 1 (12.5)
Didelphimorphia
165 31 1 (3.2)
Perissodactyla 391 109 9 (8.2)
Pilosa 6 4 1 (25.0)
Primates 253 133 4 (3.0)
Rodentia 137 103 6 (5.8) 1 (16.7)
Reptilia 345 85 0 (0) 0 (0) 0 (0) 0 (0) 0 (0)
Total 4601 1852 88 (4.8) 1(1.1) 1(1.1) 2 (2.3) 0 (0)
Genus No. Mosquitoes
Collected
No. Mosquito
Pools
No. Screened by
Flavivirus rt RT-PCR
No. Flavivirus rt RT-
PCR Positive (%)
No. Tested by ZIKV
rt RT-PCR*
No. ZIKV
rt RT-
PCR
Positive (%)
Aedes 7674 478 269 152 (56.5) 152 0 (0)
Haemagogus 67 32 11 6 (54.5) 60 (0)
Limatus 82 30 73 (42.9) 30 (0)
Culex 41223 1803 888 218 (24.5) 221 0 (0)
Uranotaenia 347 64 26 6 (23.1) 60 (0)
Psorophora 790 224 87 19 (21.8) 10 0 (0)
Coquillettidia 3070 115 94 14 (14.9) 15 0 (0)
Mansonia 2297 178 102 9 (8.8) 90 (0)
Anopheles 305 97 17 0 (0) 00 (0)
Johnbelkina 2 2 2 0 (0) 00 (0)
Sabethes 4 4 1 0 (0) 00 (0)
Trichoprosopon 8 3 2 0 (0) 00 (0)
Wyeomyia 173 12 20 (0) 00 (0)
Other 778 43 15 0 (0) 00 (0)
Total 56820 3085 1523 427 (28.0) 431 0 (0)
Figure 1: Map of the Zika Ecology Study
Field Sites in Brazil, Colombia, and Peru
Table 2. Preliminary Whole Blood PCR Results by Animal Class
Fieldwork examples
uIdentifying and sorting mosquitoes
vTakin g meas urements of a didel ph is for classif ication
wRemoving a bat from a mist net for sample collection
xConducting PRNT testing
METHODS
We conducted active ZIKV surveillance from February 2017-March 2018 in abundant
animals and mosquitoes species* at established field sites in Brazil, Colombia, and Peru
that showed:
evidence of recent or active ZIKV transmission in humans
abundance of domestic animals and wildlife
abundance and variety of mosquito species
*Our sample target was 5,000 animal samples (30 individuals per species/per study site/per campaign)
and 200,000 mosquitoes to detect a 1% seroprevalence with 95% confidence
u
v
w
Table 3. Preliminary Mosquito Pool PCR Results by Mosquito Genus
Table 1. Preliminary PRNT Results1by Animal Order
LB-5060
This research was made possible through support provided by the Office of Infectious Disease, Bureau for Global Health, U.S. Agency for International Development, under the terms of an Interagency Agreement with CDC.
Active ZIKV infection (whole blood and pool mosquitoes)
Screened by aSYBR green pan-Flavivirus real time RT-PCR
Confirmed by ZIKV specific real time RT-PCR (Lanciotti 2008)and sequencing
Previous ZIKV infection (plasma)
Tested for anti-ZIKV antibodies by plaque reduction neutralization testing (PRNT)
Assessed cross-reactivity by performing PRNT for other circulating flavivirus
Blood meal analysis (engorged mosquito abdomens)
Homogenized, extracted, and tested by conventional PCR with universal Cytb and COI primers
Sequenced product is aligned to published GenBank sequences
CONCLUSIONS
Preliminary results do not present evidence of active ZIKV circulation among the vertebrate and invertebrate
species samples, despite recent and active circulation of the virus in the neighboring human population. There is
evidence of potential ZIKV spillover, however, confirmatory testing is still in process as is the blood meal analysis
for many high interest samples like those from primates.
We anticipate final results will:
facilitate a better understanding of ZIKV’s ability to establish a sylvatic cycle outside of human transmission
devise future surveillance strategies for ZIKV in tropical ecosystems
inform public health intervention efforts
With an effort of :
~14,903 small mammal trap nights
~1,526 hours of mist netting for bats and birds
~4,339 mosquito trap nights and 424 hours of
aspirating
From 85 subsites (~14/site) classified as:
65% Peri-urban and rural village
13% Rural farm
12% Uninhabited open land and forest
10% Urban
All trapping location, sample collection,
and site characterization data were
collected on handheld android devices
using the CommCare app.
Samples were barcoded and scanned
into the electronic form for ease of data
linking.
x
We sampled from 6 field sites (2/country)
Sherman/tomahawk traps and darting techniques for
wildlife capture
CDC light/BG sentinel/resting traps and backpack/hand-
held aspirators were used to collect mosquitoes
We would like to give a huge THANK YOU to
all of our collaborating partners and
colleagues who have made this effort so
successful. There is not enough space to list
you all!
Please also visit or refer to ASTMH
presentation #217 and posters #708, #1640,
#1644, and #LB-5413 for more on this
project.
* This sample is from a female red howler monkey sampled in Santander, Colombia and is pending confirmation by viral isolation
* Includes samples that were inconclusive by screening assay
1. These data reflect number of samples with endpoint PRNT90 titers >10. All samples with titers >10 for the ZIKV PRNT went on for subsequent flavivirus PRNTs;
DENV3=Dengue virus type 3, ILHV=Ilheus virus, WNV=West Nile virus; YFV=Yellow fever virus; 2. Only Orders with ZIKV PRNT90 positive results are shown.
Laboratory analysis for the remaining 37% (4,550/12,293)
of individual and pooled samples is underway.