---
id: "edwards-2020-muscovy-ducks"
title: "Detection of Reticuloendotheliosis Virus in Muscovy Ducks, Wild Turkeys, and Chickens in Brazil"
authors:
  - "Giovana S. Caleiro"
  - "Cristina F. Nunes"
  - "Paulo R. Urbano"
  - "Karin Kirchgatter"
  - "Jansen de Araujo"
  - "Edison Luiz Durigon"
  - "Luciano M. Thomazelli"
  - "Brittany M. Stewart"
  - "Dustin C. Edwards"
  - "Camila M. Romano"
venue: "Journal of Wildlife Diseases"
year: 2020
date: "2020-07-02"
doi: "10.7589/2019-04-088"
url: "/research/publications/10-7589-2019-04-088/"
pdf: "/research/publications/10-7589-2019-04-088/dustin-edwards-10-7589-2019-04-088.pdf"
preprintDoi: "10.1101/698985"
openAccess: false
citedBy: 7
citedBySource: "OpenAlex, read 2026-09-12"
---
# Detection of Reticuloendotheliosis Virus in Muscovy Ducks, Wild Turkeys, and Chickens in Brazil

First report of reticuloendotheliosis virus in Brazil; 16.8 percent of ducks, turkeys and chickens tested were positive.

## Abstract

Reticuloendotheliosis viruses (REVs) are known to cause immunosuppressive and oncogenic disease that affects numerous avian species. Reticuloendotheliosis viruses are present worldwide and recently have been reported in South America with cases of infected commercial flocks in Argentina. We surveyed for the presence of REV in birds from a state in the northern region of Brazil using real-time PCR. We report here the presence of REV in Brazil, detected in Muscovy Ducks ( Cairina moschata ), Wild Turkeys ( Meleagris gallopavo ), and chickens ( Gallus gallus ) at a relatively high prevalence (16.8%). Phylogenetic analysis indicated a close relationship of these strains to variants in the US. This study provides evidence of REV in the Amazon biome and provides a baseline for future surveillance of the virus in the region and throughout Brazil.

## Full text

Machine-extracted from the PDF linked above. It carries the artifacts that come with reading a typeset two-column page: running heads, figure captions in the flow of the prose, and words broken across line ends. The abstract above is the registry's deposit and is the authoritative text.

SHORT COMMUNICATIONS
DOI: 10.7589/2019-04-088 Journal of Wildlife Diseases, 56(3), 2020, pp. 000–000
Ó Wildlife Disease Association 2020
Detection of Reticuloendotheliosis Virus in Muscovy Ducks, Wild
Turkeys, and Chickens in Brazil
Giovana S. Caleiro,1 Cristina F. Nunes,1 Paulo R. Urbano,1 Karin Kirchgatter,2 Jansen de Araujo,3 Edison
Luiz Durigon,3 Luciano M. Thomazelli,3 Brittany M. Stewart,4 Dustin C. Edwards,4 and Camila M.
Romano1,5 1Instituto de Medicina Tropical de Sa˜ o Paulo e Hospital das Cl´ınicas da Faculdade de Medicina,
HCFMUSP (LIM52) Av. Dr. Ene´ as Carvalho de Aguiar, 470, CEP 05403-000, Sa˜ o Paulo/SP, Brasil, Universidade de Sa˜ o
Paulo, Sa˜ o Paulo, Brazil; 2Instituto de Medicina Tropical de Sa˜ o Paulo, SUCEN, Av. Dr. Ene´ as Carvalho de Aguiar, 470,
CEP 05403-000, Sa˜ o Paulo/SP, Brazil 3 Laborat ´orio de Virologia Cl´ınica e Molecular, Instituto de Ci ˆencias Biome´ dicas
(ICB), Universidade de Sao Paulo, Av. Prof. Lineu Prestes, n8 2415CEP 05508-900, Butanta˜ , Sa˜ o Paulo/SP, Brazil;
4Department of Biological Sciences, Tarleton State University, Box T-0100, Stephenville, Texas 76402; 5Corresponding
author (email: cmromano@usp.br)
ABSTRACT: Reticuloendotheliosis retroviruses
(REVs) are known to cause immunosuppressive
and oncogenic disease that affects numerous avian
species. Reticuloendotheliosis retroviruses are
present worldwide and recently have been
reported in South America with cases of infected
commercial flocks in Argentina. We surveyed for
the presence of REV in birds from a state in the
northern region of Brazil using real-time PCR.
We report here the presence of REV in Brazil,
detected in Muscovy Ducks (Cairina moschata),
Wild Turkeys (Meleagris gallopavo), and chickens
(Gallus gallus) at a relatively high prevalence
(16.8%). Phylogenetic analysis indicated a close
relationship of these strains to variants in the
United States. This study provides evidence of
REV in the Amazon biome and provides a
baseline for future surveillance of the virus in
the region and throughout Brazil.
Key words: Avian retrovirus, Brazil, reticuloen-
dotheliosis virus, REV.
Reticuloendotheliosis viruses (REVs) are a
group of immunosuppressive and sometimes
oncogenic retroviruses that affect numerous
species of birds, including waterbirds (Anser-
iformes), game birds (Galliformes), and
perching birds (Passeriformes; Nair et al.
2013). Reticuloendotheliosis viruses has pre-
viously affected commercial poultry and has
been a recurring obstacle in the conservation
of endangered species in some countries, such
as Attwater’s Prairie Chicken (Tympanuchus
cupido attwateri; Ferro et al. 2017). In Brazil,
the impact of REV in endangered species is
not known. Among the birds that are puta-
tively susceptible to REV infection are the
critically endangered anseriform Brazilian
Merganser (Mergus octocetacius) and the
galliform Red-billed Curassow (Crax blumem-
bachii; Ministry of the Environment, 2014).
Within vaccinated flocks, REV infection can
lead to decreased efficacy of vaccinations for
avian influenza virus, Newcastle disease virus,
Marek’s disease virus (MDV), and turkey
herpesvirus due to the reduced humoral
response resulting from immunosuppression
(Sun et al. 2009). The virus was first identified
in the US in 1958, and later in China, Taiwan,
Australia, Argentina, and Canada (Singh et al.
2003; MacDonald et al. 2018). The prevalence
of REV varies from 0% to 50% depending on
the region, setting, and avian species (Jiang et
al. 2013; Ferro et al. 2017; Stewart et al.
2019).
Evidence of REV in South America was
recently demonstrated in fowlpox-vaccinated
commercial poultry flocks in Argentina (Bus-
caglia 2013). Reticuloendotheliosis virus pro-
viral DNA has been detected in the genomes
of the attenuated MDV vaccine (MD-2 strain)
and the field and vaccine strains of fowlpox
viruses (FWPV-REV; Isfort et al. 1992; Hertig
et al. 1997). Vaccine-integrated REV can be
either infectious or noninfectious (Hertig et
al. 1997; Moore et al. 2000). The Brazilian
coast serves as an important stopover site for
migratory birds coming from the northern
hemisphere. Several species migrate during
the austral winter from Argentina and Chile to
central Bolivia and Brazil (Somenzari et al.
2018). In addition, several birds, including
Passeriformes, migrate from the US, Mexico,
and Central America to northern Brazil
(Somenzari et al. 2018). However, no studies
have surveyed for the presence of REV in
1

Brazil. With more than 2,000 species of wild
birds present in the country, many of which
are endangered, determining the presence of
REV and establishing a baseline prevalence
rate could be of value for future conservation
efforts in the Amazon biome.
During 2005–2006, a total of 441 samples
were collected (blood and pooled cloacal or
orotracheal swabs) near eight different cities
in the northeastern region of Para´ state,
Brazil. Most of the samples were from
Muscovy Ducks (Cairina moschata, n¼379),
and the remaining samples were from Wild
Turkeys (Meleagris gallopavo, n¼41) and
chickens (Gallus gallus, n¼21). Orotracheal
or cloacal swab samples were obtained from a
previous avian influenza virus study (Thoma-
zelli et al. 2012). All samples were collected
from live animals with no apparent illness
using simple traps (drop nets) or manual
restraint. The birds were free-living or semi-
domesticated. The semidomesticated birds
were kept outdoors, so contact with other
birds was frequent. For the present study,
cDNA previously used in Thomazelli (2014)
was investigated for the presence of REV
genetic material. As an internal control, a
conventional PCR method for avian mito-
chondrial DNA was performed using cyto-
chrome b primers (Kocher et al. 1989). The
presence of REV proviral cDNA was detected
using primers specific to the gp90 gene (env)
and real-time PCR (Li et al. 2013). Reactions
were prepared using 2.5 mM of each primer,
7.5 lL of water, 12.5 lL of MasterMix SYBRt
Green (LifeTechnologiest, Sa˜o Paulo, SP,
Brazil), and 3 lL of purified cDNA. Plasmids
containing genes of interest were constructed
from commercially synthesized inserts (Gen-
Script, Piscataway, NJ, USA) to serve as
positive controls. In addition, partial frag-
ments of LTR-U5, Gag, and envelope from
REV-positive samples were amplified and
sequenced by the Sanger method using
previously described primers (Singh et al.
2003; Barbosa et al. 2007; Li et al. 2013). For
the envelope region, an additional primer was
designed (gp90-7242R 50-GCCAGTATGCA
CAGCCCTATCCA-30).
Of the 441 samples tested and amplified,
REV PCR products were detected in 74
samples (17%). Infected individuals included
65 Muscovy Ducks, six Wild Turkeys, and two
chickens. The REV-positive samples came from
birds in Marabitana, Vila Maracaja´, and Maraj´o.
Unfortunately, only viruses obtained from
Muscovy Ducks were successfully amplified
and sequenced. Sequencing reads were inspect-
ed for quality and consensus sequences were
built using the CLC Genomic Workbench v5
(Qiagen, Redwood City, California, USA) and
submitted to GenBank (accession numbers
MG953804–MG953809 and MN389236–
MN389242). Consensus sequences of partial
LTR/gag (808 base pairs), gag (1,168 base pairs),
and envelope (~600 base pairs) were aligned
and compared to reference strains and other
sequences, retrieved from GenBank, that rep-
resented worldwide REV (n¼16). Phylogenetic
trees were reconstructed under the maximum-
likelihood method in PhyML 3.0 software
(Guindon et al. 2010) using K80 as the best
nucleotide substitution model for all fragments
as determined by jModeltest (Darriba et al.
2012). Only sequences built with high quality
reads (five sequences from envelope region and
seven from LTR/gag—Phred .30) were used
for phylogenetic reconstructions. Together,
sequences from nine different isolates were
used to build either envelope or LTR/gag trees
or both. Genetic distance (p-distance) between
the main REV strains and one representative
Brazilian REV sample was estimated for LTR/
gag, gag, and envelope partial fragments. To
establish the phylogenetic relationships among
Brazilian and worldwide REV, phylogenetic
trees were reconstructed using LTR/gag and
envelope fragments representing Brazilian and
worldwide REV genomic sequences (n¼16; Fig.
1). Brazilian REV clustered together with
sequences sampled in the US, such as Att-
water’s Prairie Chicken reference strain APC-
566 (Barbosa et al. 2007). Genetic distance at
the nucleotide level (p-distance) between the
main REV strains and one representative
Brazilian REV (REV_2036_BR) also agreed
that Brazilian REV is more closely related to
the US samples representative of subtype 3,
such as MD-2 and APC-566, rather than to
2 JOURNAL OF WILDLIFE DISEASES, VOL. 56, NO. 3, JULY 2020

spleen necrosis virus (SNV) strain or China
isolates (Fig. 1 and Table 1).
Para´ state is located in the northern coastal
region of Brazil and has a tropical rainforest
climate. Because of its geographic location
and ecosystem, Para´ is a stopover site for
shorebirds coming from the northern hemi-
sphere during migration. In addition, several
species of migratory birds that have Argentina
and the US as their final destinations pass
through Brazil during their flight. The simi-
larity between Brazilian REV and the US
viruses suggests that the US could be the
source of REV detected in Brazil. However,
FIGURE 1. Maximum-likelihood phylogenetic reconstruction showing the relationships between reticuloen-
dotheliosis retrovirus from Brazil and other isolates. Brazilian sequences (black closed circles) are from Muscovy
Ducks (Cairina moschata). The trees were made with partial sequences from reticuloendotheliosis retrovirus env
A) and LTR/gag B) available at GenBank.
TABLE 1. Genetic identity (p-distance) in percentage at nucleotide level between Brazilian reticuloendotheliosis
retrovirus (REV) strain (REV_2036_BR) and reference strains from GenBank.
REV_2036_BR
REV reference GenBank ID LTR/gag (%) Gag (%) Env (%)
APC-566 DQ387450 99.8 100 99.8
SNV DQ003591 96 97.5 96.4
MD-2 JX912710 99.4 100 100
FPV AF246698 99.6 100 100
CSV DQ237905 NAa NA 99.5
HA9901 (China) AY842951 96 98 97.6
a NA ¼ sequence not available.
SHORT COMMUNICATIONS 3

no sequence data are available for viruses
originating in Argentina. As such, the source
of Brazilian REV cannot be definitively
determined. Representative strains of REV
include the defective REV-T, the nondefec-
tive REV-A, SNV, duck infectious anemia,
and chicken syncytial virus (CSV), and the
MD-2 and FWPV vaccine-integrated strains.
As there is relatively little genetic variation
among REV strains, it is difficult to determine
the origin and classification of novel isolates
(Bohls et al. 2006). In our analysis, Brazilian
REV differs only 0.0–0.5% from both FWPV
and MD-2 integrated REVs and nonintegrat-
ed APC-566 and CSV. We inspected for point
mutations, which have been characterized as
specific for FWPV-REV (Tadese et al. 2008).
According to the genetic pattern observed, the
Brazilian REV samples are all non–FWPV-
integrated strains. However, not all FWPV-
REV envelope sequences available on Gen-
Bank (KY498002, AF246698, and JX217830)
contain this signature, indicating that some
genetic variability among integrated strains
exists and that determining the integration
status based on a few point mutations is
inaccurate (Tadese et al. 2008). Therefore, it
remains unknown if Brazilian REV is present
as infectious particles or is instead integrated
within a large DNA virus genome.
Reticuloendotheliosis viruses can infect a
number of species, including captive and wild
perching birds, game birds, and waterbirds.
Although we detected REV in Muscovy
Ducks, Wild Turkeys, and chickens, we could
amplify and sequence the virus only from
ducks. It is possible that REV viral loads were
lower in other species, precluding our ability
to generate large amplicons. Muscovy Ducks
are native to Mexico and Central and South
America, but populations of Muscovy Ducks
also reside in the US, mainly in Florida and
southern Texas. These birds are essentially
nonmigratory or irregular migrants without
established migration patterns, only migrating
short distances to avoid dry weather and
fluctuating water conditions. Chickens and
turkeys, which are frequently associated with
REV infections, are also nonmigratory birds.
Therefore, we consider it unlikely that the
positive birds found in Brazil were infected
elsewhere.
Here, we describe the presence of REV in
Brazil and present sequences of REV provirus
from South America. The REV found in Brazil
is similar to other common circulating strains,
including those found in the US. Additionally,
considering that REV was present in samples
from three of the five regions collected from
Para´ in 2005–2006 (located 200–500 km apart
from each other), it is very likely that the virus
has spread to other states in Brazil. We
hypothesize that the virus could be carried
by migratory birds that stop over in the
northern part of the country on their way to
and from North America. These results
suggest the need for additional studies to
further determine the prevalence and genetic
variability of REV in Brazilian wild and
captive birds and also to perform risk
assessment studies dedicated to free-range
and captive commercial avian species. In
addition to REV, it will be also important to
survey for the presence of lymphoproliferative
disease virus in the future, as it is an
oncogenic retrovirus that infects domestic
and wild turkeys and chickens across the US
(Alger et al. 2017).
All procedures involving wild and captive
birds were approved by the Animal Ethics
Committee from the Instituto de Medicina
Tropical de Sa˜o Paulo under protocol 000283A,
and licensed by the Ministe´ rio do Meio
Ambiente–MMA at the Instituto Chico Men-
des de Conservaca˜o da Biodiversidade (ICM-
Bio/SISBIO) under protocols 34605-7 45527-2,
54616-1, and 201/2006 CGFAU. This work was
supported by Fundac¸a˜o de Amparo a Pesquisa
do Estado de Sa˜o Paulo (FAPESP Project
#2015/05958-3) and by the Programa de
fomento `as atividades de lideranc¸as cient´ıficas
dos LIMs do Hospital das Cl´ınicas da FMUSP
(PROFAP-LIM/HCFMUSP) number 20/2019.
G. Caleiro holds a CAPES scholarship.
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SHORT COMMUNICATIONS 5