Am. J. Trop. Med. Hyg., 87(6), 2012, pp. 1132–1139
Copyright © 2012 by The American Society of Tropical Medicine and Hygiene
Orthobunyaviruses, a Common Cause of Infection of Livestock in the Yucatan
Peninsula of Mexico
Bradley J. Blitvich,* Rungrat Saiyasombat, Amelia Travassos da Rosa, Robert B. Tesh, Charles H. Calisher,
Julian E. Garcia-Rejon, Jose ´ A. Farfa ´n-Ale, Rube ´n E. Loron ˜o, Arturo Bates, and Maria A. Loron ˜o-Pino
Department of Veterinary Microbiology and Preventive Medicine, College of Veterinary Medicine, Iowa State University, Ames, Iowa;
Center for Biodefense and Emerging Infectious Diseases, Department of Pathology, University of Texas Medical Branch, Galveston,
Texas; Arthropod-Borne and Infectious Diseases Laboratory, Department of Microbiology, Immunology and Pathology, College of
Veterinary Medicine and Biomedical Sciences, Colorado State University, Fort Collins, Colorado; Laboratorio de Arbovirologia,
Universidad Autonoma de Yucatan, Merida, Yucatan, Mexico; Oasis, Cholul, Me ´rida, Yucata ´n, Me ´xico; Centro Me ´dico
Veterinario del Oriente, Tizimı ´n, Yucata ´n, Me ´xico
Mexico, a serologic investigation was performed using serum samples from 256 domestic animals (182 horses, 31 sheep,
1 dog, 37 chickens, and 5 turkeys). All serum samples were examined by plaque reduction neutralization test using
Cache Valley virus (CVV), Cholul virus (CHLV), South River virus (SOURV), Kairi virus, Maguari virus, and
Wyeomyia virus. Of the 182 horses, 60 (33.0%) were seropositive for CHLV, 48 (26.4%) were seropositive for CVV,
1 (0.5%) was seropositive for SOURV, 60 (33.0%) had antibodies to an undetermined orthobunyavirus, and 13 (7.1%)
were negative for orthobunyavirus-specific antibody. Of the 31 sheep, 6 (19.3%) were seropositive for CHLV, 3 (9.7%)
were seropositive for CVV, 4 (12.9%) were seropositive for SOURV, 16 (51.6%) had antibodies to an undetermined
orthobunyavirus, and 2 (6.5%) were negative for orthobunyavirus-specific antibody. The single dog was seropositive for
SOURV. Four (11%) chickens had antibodies to an undetermined orthobunyavirus, and 1 (20%) turkey was sero-
positive for CHLV. These data indicate that orthobunyaviruses commonly infect livestock in the Yucatan Peninsula.
To determine the seroprevalence of selected orthobunyaviruses in livestock in the Yucatan Peninsula of
The family Bunyaviridae comprises the largest group of
arthropod-borne viruses (arboviruses) and consists of five
genera: Orthobunyavirus, Phlebovirus, Hantavirus, Nairovirus,
and Tospovirus.1,2A characteristic feature of all viruses in the
family Bunyaviridae is that they possess a tripartite, single-
stranded, negative-sense RNA genome.2,3The three genomic
segments are designated as small (S), medium (M), and large
(L). The genus Orthobunyavirus contains 18 serogroups,
including the Bunyamwera (BUN) and California (CAL)
serogroups. Viruses in the BUN serogroup include Cache Val-
ley virus (CVV), Cholulvirus (CHLV)and Kairi virus (KRIV).
The CAL serogroup includes South River virus (SOURV), as
well as important human pathogens such as La Crosse,
Jamestown Canyon and Tahyna viruses.
We recently reported the isolation of 20 orthobunyaviruses
from mosquitoes in the Yucatan Peninsula of Mexico in 2007
and 2008.4–6These isolates were identified as CVV (n = 17),
CHLV (n = 1), KRIV (n = 1), and SOURV (n = 1). Cache
Valley virus is the best characterized of these four viruses.
The initial isolation of CVV was made from Culiseta inornata
mosquitoes in Utah in 1956 and the virus, or subtypes of it,
have since been detected across much of the United States as
well as Canada, Mexico, Panama, Ecuador, and Jamaica.6–12
Cache Valley virus has been associated with two cases of
severe human disease in the United States, the first of which
occurred in North Carolina in 1995 and the second in Wisconsin
in 2003.13,14In addition, Fort Sherman virus, an antigenic
subtype of CVV, was responsible for a human case of febrile
illness in Panama in 1985.9
Cache Valley virus is also a pathogen of ungulates, and
CVV infections in sheep are common and can result in embry-
onic and fetal death, stillbirths, and multiple congenital
defects.15–18This virus has also been isolated from a sick
caribou and an apparently healthy horse and cow, and anti-
bodies to this virus have been detected in a variety of other
vertebrates including deer, elk, goats, and pigs.18–22The sero-
prevalence for CVV in white-tailed deer in disease-endemic
areas of the United States is often high and usually exceeds
70%.21,23,24In this region, white-tailed deer have been impli-
cated as the natural reservoir host of CVV.21
Sequence and phylogenetic data indicate that CHLV is
most likely a natural reassortant that acquired its S RNA
segment from CVV and its M and L RNA segments from
Potosi virus (POTV).4A single isolation of this virus has been
made from a pool of Ochlerotatus (Aedes) taeniorhynchus
collected in Merida in the Yucatan Peninsula in 2007.4,5The
natural reservoir host(s) of CHLV has not been determined,
and it is not known whether this virus is a pathogen of humans
or other vertebrates. Potosi virus, the M and L segment donor
of CHLV, has been identified in several states in the eastern
and central United States, including Texas, although it could
also be present in Mexico because it is one of the precur-
sor viruses of CHLV25–28(Tesh R, Travassos da Rosa A,
unpublished data). Potosi virus is not a recognized pathogen
of humans or other vertebrates. The natural reservoir host of
POTV is also suspected to be white-tailed deer.21
Kairi virus was originally isolated from mosquitoes in
Trinidad in 1955, and later was isolated from mosquitoes
and wild vertebrates in Brazil, mosquitoes in Colombia,
and a febrile horse in Argentina.29–32More recently, a single
isolation of KRIV was made from a pool of Oc. taeniorhynchus
collected in Merida in 2007.5,33Antibodies to KRIV were
detected in 5% of humans sampled in Argentina in 2004 and
2005.34In addition, antibodies that neutralized KRIV were
identified in 48% of horses sampled in Argentina in 1983
*Address correspondence to Bradley J. Blitvich, Department of
Veterinary Microbiology and Preventive Medicine, Iowa State
University, 2116 Veterinary Medicine, Ames, IA 50011. E-mail:
Two other members of the BUN serogroup known to be
present in Latin America are Maguari virus (MAGV) and
Wyeomyia virus (WYOV), although neither has been
reported in the Yucatan Peninsula. Maguari virus has been
isolated from mosquitoes, and antibodies to this virus have
been detected in humans, horses, sheep, and cattle in various
parts of South America and the Caribbean islands.36–38
Wyeomyia virus has been isolated from mosquitoes and a
human, and antibodies to this virus have been detected
in humans in Central America, South America, and the
As noted earlier, SOURV belongs to the CAL serogroup.
This poorly characterized virus was originally isolated from
mosquitoes in New Jersey in 196040and later from mosqui-
toes in Pennsylvania,41Georgia (Mead DG, unpublished
data) and the Yucatan Peninsula.6The SOURV isolate from
Mexico is genetically and serologically distinct from the pro-
totype strain and represents a novel subtype of SOURV.42
Until now, there have been no published studies that reported
detection of antibodies to SORV in vertebrates. Conse-
quently, the host range of this virus has not been defined.
The International Committee on Taxonomy of Viruses has
assigned the acronym of SORV to this virus, but we use
SOURV in this article because the acronym SORV is also
used for Sororoca virus, which was discovered first.
There is no recent information on the seroprevalence of
orthobunyaviruses in vertebrates in the Yucatan Peninsula.
Therefore, the overall goal of this study was to determine the
seroprevalence of orthobunyaviruses in livestock in this region.
To achieve this goal, an archived collection of serum samples
from various species of domestic vertebrates were assayed by
plaque reduction neutralization tests (PRNTs) using the four
orthobunyaviruses (CVV, CHLV, KRIV, and SOURV) iso-
lated during recent entomologic investigations in the Yucatan
Peninsula, as well as two other orthobunyaviruses (MAGV and
WYOV) known to be present in Central and South America.
MATERIALS AND METHODS
Description of study sites. Domestic animals were sampled
in 26 study sites located in 5 municipalities (Figure 1). Four
municipalities (Panaba, Tizimin, Merida, and Tzucacab) are
in Yucatan State and one (Jose Maria Morelos) is in Quintana
Roo State. Yucatan and Quintana Roo are two of the three
states that comprise the Yucatan Peninsula of Mexico. All
study sites were on privately owned ranches or farms. The
climate and topography of the study sites are similar. The
climate is tropical. The average annual rainfall in each study
site ranges from 600 to 1,100 mm, the average annual temper-
ature is 26°C, and the average elevation is < 20 meters.
Sample population and serum collections. Blood samples
were obtained from horses (n = 182), sheep (n = 31), chickens
(n = 37), turkeys (n = 5) and a dog (n = 1) from September
2007 through October 2008. The horses were from the munici-
palities of Panaba (n = 108), Tizimin (n = 63), and Jose Maria
Morelos (n = 11). The sheep, dog, and chickens were from
Merida, and the turkeys were from Tzucacab. According to
the owners, none of the animals had ever been outside the
Yucatan Peninsula. All animals were regularly monitored
(usually daily) by their caregivers for signs of illness. Six
horses had clinical signs at the time of sampling (fever, ataxia,
lethargy, depression, paralysis, and/or encephalitis); all other
animals appeared healthy. The age range of the horses was
8 months to 15 years, and the mean age was 6.7 years. The age
range of the sheep was 4 months to 6 years, and the mean age
was 17 months. Ages of the dog, chickens, and turkeys were
Plaque reduction neutralization tests. The PRNTs were
conducted according to standard methods43using CHLV
(strain CHLV-Mex07), CVV (strain CVV-478), KRIV (strain
KRIV-Mex07), SOURV (strains SORV-252 and NJO-94f),
MAGV (strain BeAr7272) and WYOV (strain prototype).
South River virus (strain NJO-94f), MAGV, and WYOV
were obtained from the World Arbovirus Reference Collec-
tion at the University of Texas Medical Branch in Galveston,
Texas. The SOURV strain NJO-94f was originally isolated
from mosquitoes in New Jersey in 1960.40The MAGV strain
BrAr7272 was obtained from mosquitoes in Brazil in 1957.29
The WYOV prototype was originally isolated from mosqui-
toes in Colombia in 1940.44All other viruses were isolated
during our previous entomologic investigations in the Yucatan
Peninsula and have been described elsewhere.4–6
The PRNTs were performed using African green monkey
kidney (Vero) cells. Initially, all serum samples were screened
at a single dilution of 1:20. Serum samples that tested positive
for antibodies to any of these viruses were further diluted and
tested by PRNT to determine their end-point titers. Titers
were expressed as the reciprocal of highest serum dilutions
yielding ³ 90% reduction in the number of plaques (PRNT90).
For etiologic diagnosis, the PRNT90 antibody titer to the
respective virus was required to be at least four-fold greater
than that to the other viruses tested. The exception to this rule
was when the PRNT90titers for two or more virus species
were ³ 1,280. In such instances, the animal was suspected
to have had at least two orthobunyavirus infections but was
assigned the conservative diagnosis of seropositive to an
undetermined orthobunyavirus(es) to avoid potential mis-
diagnosis because antibody responses in vertebrates sequen-
tially infected with orthobunyaviruses are not well understood.
There is only one report that describes the antibody responses
in vertebrates experimentally inoculated with two different
orthobunyaviruses21and, to the best of our knowledge, high
PRNT titers have not been reported in vertebrates in Mexico.
Complement fixation tests. Complement fixation (CF) tests
were performed to determine whether this technique can
differentiate between antibodies to CHLV and POTV. The
PRNT cannot be used for such purposes because these
Yucatan Peninsula of Mexico from which livestock were sampled.
Geographic locations of the five municipalities in the
ORTHOBUNYAVIRUSES IN MEXICAN LIVESTOCK
two viruses share the same M RNA segment and therefore
their surface glycoproteins are antigenically indistinguishable.
However, complement-fixing antigenic determinants are
associated with the S segment–encoded nucleocapsid protein.
The CF tests were performed using a microtiter technique
with two full units of guinea pig complement.43Titers were
recorded as the highest dilutions giving 3+ or 4+ fixation of
complement on a scale of 0 to 4+. Viral antigens for the CF
test were prepared from newborn mouse brains that had
been inoculated with CHLV (strain CHLV-Mex07) or POTV
(89-3380). We also included CVV (strain Holden) in these
experiments. Immune serum samples for the CF test were
prepared in adult mice that had been inoculated with 10%
suspensions of infected suckling mouse brain of CHLV,
CVV, or POTV. The immunization schedule consisted of four
intraperitoneal injections of suspensions mixed with the com-
plete Freund’s adjuvant given at weekly intervals.
Isolation of RNA and reverse transcription polymerase
chain reactions. Total RNA was extracted from serum sam-
ples of all symptomatic livestock using the QIAamp viral
RNA extraction kit (QIAGEN, Valencia, CA) and analyzed
by reverse transcription polymerase chain reaction using
orthobunyavirus-reactive and CHLV-reactive primers. The
orthobunyavirus-specific primers, BCS82 (5¢-ATG ACT
GAG TTG GAG TTT CAT GAT GT-3¢) and BCS332V (5¢-
TGT TCC TGT TGC CAG GAA AAT-3¢), are specific for a
251-nucleotide region of the S RNA segment.45The CHLV-
reactive primers, CHLV-M1488-F (5¢-TGA TAC TGG CAG
CAG CAG AGA CAG-3¢) and CHLV-M1870-R (5¢-GGC
TGT TAG AAT GCC TTG CAC ATG-3¢), are specific for a
387-nucleotide region of the M RNA segment. Comple-
mentary DNAs were generated using Superscript III reverse
transcriptase (Invitrogen, Carlsbad, CA), and PCRs were
performed using Taq polymerase (Invitrogen) according to
the manufacturer’s instructions.
Seroprevalence of orthobunyaviruses in horses. Antibodies
to one or more orthobunyaviruses were detected by PRNT in
serum samples from 169 (92.9%) of 182 horses. Of these
horses, 60 (33.0%) were seropositive for CHLV, 48 (26.4%)
were seropositive for CVV, 1 (0.5%) was seropositive for
SOURV, and 60 (33.0%) had antibodies to an undetermined
orthobunyavirus (Table 1). The CHLV-seropositive horses
had CHLV PRNT90titers of 40 (n = 1), 80 (n = 2), 160 (n = 5),
320 (n = 6), 640 (n = 17), 1,280 (n = 12), 2,560 (n = 15), and
5,120 (n = 2). The CVV-seropositive horses had CVV
PRNT90titers of 80 (n = 1), 160 (n = 3), 320 (n = 12), 640
(n = 19), 1,280 (n = 10), 2,560 (n = 2), and 5,120 (n = 1). The
SOURV-seropositive horse had a PRNT90titer of 320 when
NJO-94f (the SOURV isolate from New Jersey) was used
(H-133 in Table 2). Interestingly, the PRNT90titer for this
horse was eight-fold lower when the SOURV isolate from
Mexico was used in the PRNT analysis. Five of the 60 horses
seropositive for an undetermined orthobunyavirus(es) had
PRNT90titers ³ 1,280 for at least two orthobunyaviruses
(CHLV, CVV, and/or KRIV). Of the remaining 55 horses
with antibodies to an undetermined orthobunyavirus(es),
the PRNT90titer was usually highest when CHLV or CVV
was used in the PRNT analysis and often there was a two-fold
difference between the highest and second highest titer. Repre-
sentative PRNT data from 12 horses with antibodies to
orthobunyaviruses are shown in Table 2.
The seroprevalence of orthobunyaviruses in horses in all
three municipalities was high. Antibodies to orthobunyaviruses
were detected in 58 (92.1%) of 63 horses in Tizimin,
100 (92.6%) of 108 horses in Panaba, and 11 (100%) of
11 horses in Jose Maria Morelos. Of the 63 horses sampled in
Tizimin, 22 (34.9%) were seropositive for CHLV, 14 (22.2%)
were seropositive for CVV, 21 (33.3%) had antibodies to an
undetermined orthobunyavirus, 1 (1.6%) was seropositive for
SOURV, and 5 (7.9%) were negative for orthobunyavirus-
specific antibody. Of the 108 horses sampled in Panaba,
33 (30.6%) were seropositive for CHLV, 32 (29.6%) were
seropositive for CVV, 35 (32.4%) had antibodies to an
undetermined orthobunyavirus, and 8 (7.4%) were negative
for orthobunyavirus-specific antibody. Of the 11 horses sam-
pled in Jose Maria Morelos, 5 (45.5%) were seropositive for
CHLV, 2 (18.2%) were seropositive for CVV, and 4 (36.4%)
had antibodies to an undetermined orthobunyavirus.
The age range of the 169 horses with orthobunyavirus-specific
antibody was 8 months to 15 years, and the mean age was
7.0 years. The age range of the 13 horses negative for
orthobunyavirus-specific antibody was 12 months to 6 years,
and the mean age was 2.4 years. Six horses had clinical signs at
the time of sampling. Of these horses, two were seropositive
for CHLV, three were seropositive for an undetermined
orthobunyavirus, and one was negative for orthobunyavirus-
specific antibody. Viral RNA was not detected in the serum
of any symptomatic horses by reverse transcription poly-
merase chain reaction using orthobunyavirus or CHLV-
Seroprevalence of orthobunyaviruses in sheep. Antibodies
to orthobunyaviruses were detected by PRNT in serum sam-
ples from 29 (93.5%) of 31 sheep. Of these sheep, 6 (19.3%)
were seropositive for CHLV, 3 (9.7%) were seropositive for
CVV, 4 (12.9%) were seropositive for SOURV, 16 (51.6%)
had antibodies to an undetermined orthobunyavirus, and
2 (6.5%) were negative for orthobunyavirus-specific anti-
bodies (Table 1). The CHLV-seropositive sheep had CHLV
PRNT90 titers of 80 (n = 1), 160 (n = 1), 1,280 (n = 2),
Seroprevalence of orthobunyavirus neutralizing antibodies in livestock in Yucatan Peninsula of Mexico*
No. (%) of livestock seropositive for each orthobunyavirus
*CHLV = Cholul virus; CVV = Cache Valley virus; SOURV = South River virus; KRIV = Kairi virus; MAGV = Maguari virus; WYOV = Wyeomyia virus.
†Undetermined orthobunyavirus (see text).
BLITVICH AND OTHERS
2,560 (n = 1), and 5,120 (n = 1). The CVV-seropositive sheep
hadCVVPRNT90titersof40(n= 1),160(n= 1),and1,280(n=
1). The SOURV-seropositive sheep all had SOURV PRNT90
titers of 640 when strain NJO-94f was used. In contrast, the
PRNT90titers for these four sheep ranged from 20 to 80 when
SORV-252 was used. Of the 16 sheep seropositive for an
undetermined orthobunyavirus(es), the PRNT90titer was usu-
ally highest when CHLV was used for the PRNT analysis and
often there was a two-fold difference between the highest and
specific antibody were five and seven months of age, and the
mean age of the sheep seropositive for orthobunyaviruses was
17.7 months. Representative PRNT data from eight sheep
with antibodies to orthobunyaviruses are shown in Table 2.
Seroprevalence of orthobunyaviruses in other vertebrates.
The single dog was seropositive for SOURV (Table 1). As
observed with the horses and sheep, the highest SOURV titer
occurred when the PRNTs were performed with NJO-94f
(D-194 in Table 2). Four (10.8%) of the 37 chickens had anti-
bodies to an undetermined orthobunyavirus(es). All four of
these chickens had KRIV PRNT90titers of 20, and the titers
for all other viruses tested were < 20. Antibodies to CHLV
were detected in 1 (20.0%) of the 5 turkeys. The CHLV
PRNT90titer for this bird was 80 (T-005 in Table 2).
Complement fixation tests. The CF tests were performed
using antisera and antigens from mice that had been experi-
mentally inoculated with CHLV, CVV, or POTV. All antisera
gave indistinguishable titers (less than a four-fold change in
both directions), indicating that this test cannot be used to dif-
ferentiate between antibodies to these three viruses (Table 3).
Therefore, this technique was not used to further analyze serum
samples from livestock in the Yucatan Peninsula.
We provide serologic evidence that orthobunyaviruses
commonly infect livestock in the Yucatan Peninsula. Anti-
bodies to orthobunyaviruses were identified in all five verte-
brate species examined and orthobunyavirus activity was
detected in all five municipalities represented in this study.
The seroprevalence for orthobunyaviruses in horses and
sheep was particularly high (approximately 93%) and at least
three viruses (CHLV, CVV, and SOURV) were shown to be
responsible for these infections. There is no other published
information on the host range of the recently described
CHLV or the poorly characterized SOURV, nor are there
data on the seroprevalence of these two viruses in vertebrates
in other geographic regions. A number of serologic investiga-
tions have determined the seroprevalence of CVV in various
vertebrates but these studies have mostly been confined to the
United States.21–23,37,46–51For instance, 19% of sheep sampled
in Texas in 1981 were seropositive for CVV.46In another
study, antibodies that neutralized CVV were detected in
84 (95%) of 88 horses, 61 (52%) of 118 cattle, and 6 (27%) of
Plaque reduction neutralization test results for a subset of livestock with antibodies to orthobunyaviruses, Yucatan Peninsula of Mexico*
PRNT diagnosisCHLV CVVKRIV
MAGV WYOVNJO-94f SORV-252
*PRNT = plaque reduction neutralization test; CHLV = Cholul virus; CVV = Cache Valley virus; SOURV = South River virus; KRIV = Kairi virus; MAGV = Maguari virus; WYOV =
Wyeomyia virus; – = < 20.
†Sample IDs beginning with a H, S, D, C, and T denote serum samples from horses, sheep, dogs, chickens, and turkeys, respectively.
‡Two SOURV isolates (NJO-94f and SORV-252) were used in the PRNT analysis.
Results of complement fixation tests performed with Cholul, Potosi
and Cache Valley viruses*
Immune serum (antibodies)
Cholul virusPotosi virus Cache Valley virus
Cache Valley virus
*Values refer to the highest dilution of antiserum with complement fixation activity.
ORTHOBUNYAVIRUSES IN MEXICAN LIVESTOCK
22 sheep in Virginia and Maryland during 1957–1961.22Neu-
tralizing antibodies to CVV were also detected in 100 (72%)
of 138 white-tailed deer in Minnesota in 1988 and 1989.23
Therefore, the moderately high seroprevalence for CVV in
sheep and horses (10% and 27%, respectively) and the high
overall seroprevalence for orthobunyaviruses in these verte-
brate species (approximately 93%) in the Yucatan Peninsula
are not dissimilar to the seroprevalences reported for ungu-
lates sampled in other serologic investigations. However,
one important aspect of this study is that it provides recent
information on the seroprevalence of orthobunyaviruses in
vertebrates in Mexico.
Cholul virus and POTV share the same M RNA segment.
Therefore, because their surface glycoproteins are antigeni-
cally indistinguishable, antibodies to these proteins cannot be
differentiated by PRNT. Although complement-fixing anti-
genic determinants are associated with the S segment–encoded
nucleocapsid protein, the CF test was also unable to differenti-
ate between antibodies to CHLV and POTV. Thus, we cannot
dismiss the possibility that POTV was the cause of infection in
some or all of the 67 animals (60 horses, 6 sheep, and 1 turkey)
considered to be seropositive for CHLV. However, we con-
sider it more likely that the aforementioned animals had been
infected with CHLV because this virus has been isolated in the
Yucatan Peninsula, and there is no direct evidence that POTV
is present in this region. It is noteworthy that serum samples
from the six sheep considered to be seropositive for CHLV
were collected in Merida in 2008. The pool of mosquitoes
yielding CHLV in our study was collected in Merida less than
12 months earlier.4,5Nevertheless, we cannot dismiss the possi-
bility that POTV was responsible for some or all of these
infections and that a more accurate PRNT diagnosis for the
aforementioned animals could be seropositive for CHLV or
POTV or seropositive for CHLV or a CHLV-like virus.
Five horses and two sheep had PRNT90titers ³ 1,280 for at
least two orthobunyaviruses. We believe that this is the first
report of high PRNT titers in vertebrates in Mexico with
naturally acquired orthobunyavirus infections. High antibody
titers are often reported in flavivirus serologic investigations
performed in geographic areas where multiple flaviviruses
circulate and have been attributed to exposure to two or more
flaviviruses.52–54For example, all four dengue flaviviruses are
present in Mexico and patients in this region often have high
PRNT titers to all serotypes.53It seems likely that the afore-
mentioned horses and sheep had infections with at least two
orthobunyaviruses and that the responses we detected may
have been anamnestic responses. In this regard, Blackmore
and Grimstad reported high neutralizing antibody titers to
CVV in white-tailed deer experimentally inoculated with
CVV, then POTV.21The mean ± SE reciprocal antibody titer
for CVV by virus neutralization assay was 839 ± 228 at seven
days post-inoculation with the secondary virus. This study is
the only one to describe antibody responses in vertebrates
Because the antibody responses in vertebrates with secondary
orthobunyavirus infections are poorly understood as com-
pared with secondary flavivirus infections, we have therefore
interpreted our PRNT data with caution and have assigned the
conservative diagnosis of seropositive for an undetermined
orthobunyavirus(es) to avoid potential misdiagnosis. None-
theless, it is feasible that these animals have been infected
with two or more orthobunyaviruses and that secondary
orthobunyavirus infection could be a more accurate diagnosis.
Alternatively, these animals may have produced unusually
high neutralizing antibody titers after exposure to a single
orthobunyavirus.If this is the case,these animals are seropositive
for CHLV (three horses and two sheep), CVV (one horse), and
an undetermined orthobunyavirus (one horse). For instance,
horse H-139 has CHLV, CVV, and KRIV PRNT90titers of
5,120, 1,280 and 1,280, respectively (Table 2) and therefore
could be considered seropositive for CHLV.
A high proportion of horses (33%) and sheep (52%) had
antibodies to an undetermined orthobunyavirus(es). One
explanation for this finding is that many of these animals had
been exposed to two or more orthobunyaviruses, thus making
it difficult to make a definitive diagnosis. If this hypothesis is
correct, we consider it most likely that the orthobunyaviruses
responsible for these dual infections are CHLV and CVV
because these two viruses were the most common causes of
infection in this study. However, in our studies, many animals
seropositive for an undetermined orthobunyavirus did not
have extremely high PRNT90titers. For example, the highest
PRNT90titer for 41 of the 60 horses with antibodies to an
undetermined orthobunyaviruses did not exceed 320. This
observation does not necessarily refute our hypothesis.
Blackmore and Grimstad reported data that imply that high
neutralizing antibody titers are not always a consequence of
sequential orthobunyavirus infections.21Seven days after
inoculation with the second virus, the mean ± SE reciprocal
antibody titers in deer sequentially inoculated with POTV
followed by CVV were 206 ± 60 and 96 ± 16, respectively. It
is also important to note the approximately two-fold differ-
ence in mean antibody titers in these deer because many of
the horses and sheep with undetermined orthobunyavirus
infections also exhibited a two-fold difference between their
highest and second highest PRNT titer.
Another explanation for the high proportion of horses and
sheep with antibodies to an undetermined orthobunyavirus(es)
is that some of these animals had been infected with an
orthobunyavirus not included in the PRNT analysis. How-
ever, our PRNTs were not restricted to orthobunyaviruses
known to be present in the Yucatan Peninsula. Two addi-
tional orthobunyaviruses (MAGV and WYOV), which have
been reported elsewhere in Latin America and can infect
some of the animal species we studied were included. Never-
theless, a subset of animals may have been infected with
another orthobunyavirus such as Northway, Tensaw, or Main
Drain viruses. Although these viruses have not been reported
in Mexico, they have been associated with livestock infections
in the United States.47,55For instance, antibodies to Northway
virus were identified in 44% of horses sampled in California
during 1968–1972.55The establishment of a continuous
entomologic-based arbovirus surveillance program in the
Yucatan Peninsula would enable identification of other
orthobunyaviruses that may be present in this region.
Six horses had signs of illness at the time of sampling, includ-
ing two horses (H-2 and H-265) that were seropositive for
CHLV. Horse H-2 had neurologic signs (facial paralysis and
encephalitis) and later died, and horse H-265 exhibited pos-
terior ataxia. Three of the other symptomatic horses (H-116,
H-263, and H-264) were seropositive for an undetermined
orthobunyavirus. Horse H-116 exhibited lethargy, horse H-263
had a fever and posterior ataxia and later died, and horse
H-264 had posterior ataxia. The remaining horse was negative
BLITVICH AND OTHERS
for orthobunyavirus-specific antibody. It was not known
whether the clinical signs in horses H-2 and H-265 were a result
of CHLV infection, but we speculate that this was not the case
because CVV and POTV, the two precursor viruses of CHLV,
are not recognized equine pathogens. An IgM enzyme-linked
immunosorbent assay has not been developed for any BUN
serogroup virus, such an assay would be a significant advance
in orthobunyavirus surveillance studies because it would
enable detection of acute infections. The PRNTs can be used
to identify recent orthobunyavirus infections when paired
acute-phase and convalescent-phase serum samples are avail-
able, but for our studies only single serum samples were avail-
able from each animal.
Antibodies to CHLV and an undetermined KRIV-like
virus were identified in 1 (20%) of 5 turkeys and 4 (10.8%)
of 37 chickens, respectively. Several other studies also
describe the identification of antibodies to BUN serogroup
viruses in birds.56–58For instance, antibodies that neutralized
MAGV were detected in 69 (10.6%) of 649 free-ranging birds
of various species in Argentina in 2004 and 2005.56Our PRNT
data indicate that the seroprevalence for orthobunyaviruses in
domestic birds is much lower compared with seroprevalences
in mammals in the Yucatan Peninsula. One explanation for
this finding is that the major vectors of orthobunyaviruses in
this region have a preference for mammalian blood. In this
regard, all the orthobunyaviruses isolated in our recent
entomologic investigations in the Yucatan Peninsula were
obtained from Oc. taeniorhynchus.5,6Although the host-feeding
preference of Oc. taeniorhynchus in the Yucatan Peninsula
has not been determined, Oc. taeniorhynchus in other regions
of North America have been shown to feed almost exclusively
on large mammals.59–61
Antibodies to SOURV were identified in 4 (12.9%) of
31 sheep, 1 (0.5%) of 182 horses, and the single dog sampled
in this study. Surprisingly, the PRNT90titers of these animals
were always greater when the PRNTs were performed with
NJO-94f, the SOURV isolate collected in New Jersey in 1960,
compared with SORV-252, which was isolated from mosqui-
toes in the Yucatan Peninsula in 2008.6,40The NJO-94f
PRNT90 titers were usually 8–16-fold greater than their
corresponding SORV-252 PRNT90titers and this finding is
also surprising because such differences are more indicative
of distinct viral species than subtypes. We recently demon-
strated by cross-PRNT using serum samples from mice inocu-
lated with SORV-252 and NJO-94f, and these isolates are
distinct subtypes of SOURV.42The contrasting serologic find-
ings between the two studies could be caused by differences
in the antibody responses of rodents and larger mammals
after SOURV infection or to the duration of time between virus
infection and serum collection. Although unlikely, the consis-
tently higher NJO-94f PRNT90 titers compared with the
corresponding SORV-252 PRNT90titers could be the result of
the NJO-94f subtype also circulating in the Yucatan Peninsula,
although it has not yet been found there.
In summary,we provide
orthobunyaviruses commonly infect livestock in the Yucatan
Peninsula. It is not known whether these viruses are also
responsible for morbidity and mortality in livestock in this
region, but future research is necessary to address this issue.
The high seroprevalence for orthobunyaviruses in mammals
also implies that the major orthobunyavirus vectors in the
Yucatan Peninsula have a strong preference for mammalian
blood. It is therefore important that the potential impact of
orthobunyaviruses on human health in the Yucatan Peninsula
be determined. This is especially true for CVV because this
virus is a recognized pathogen of humans.
Received March 23, 2012. Accepted for publication August 25, 2012.
Published online October 8, 2012.
Financial support: This study was supported by the Iowa State
University Plant Sciences Institute Virus-Insect Interactions Initia-
tive and in part by grant 5R21AI067281-02 from the U.S. National
Institutes of Health. Amelia Travassos da Rosa and Robert B. Tesh
were supportedby National
Institutesof Health contract
Authors’ addresses: Bradley J. Blitvich and Rungrat Saiyasombat,
Veterinary Medicine, Department of Veterinary Microbiology and
Preventive Medicine, Iowa State University, Ames, IA, E-mails:
email@example.com and firstname.lastname@example.org. Amelia Travassos da
Rosa and Robert B. Tesh, Center for Biodefense and Emerging Infec-
tious Diseases, Department of Pathology, University of Texas Medical
Branch, Galveston, TX, E-mails: email@example.com and rtesh@utmb
.edu. Charles H. Calisher, Arthropod-Borne and Infectious Diseases
Laboratory,Department of Microbiology, Immunology and Pathology,
College of Veterinary Medicine and Biomedical Sciences, Colorado
State University, Fort Collins, CO, E-mail: firstname.lastname@example.org.
Julian E. Garcia-Rejon, Jose ´ A. Farfa ´n-Ale, and Maria A. Loron ˜o-
Pino, Laboratorio de Arbovirologia, Centro de Investigaciones
Regionales Dr. Hideyo Noguchi, Universidad Autonoma de Yucatan,
Av. Itzaes No. 490+59, Centro, Merida, Yucatan, Mexico, E-mails:
email@example.com, firstname.lastname@example.org, and maria.lorono@gmail
.com. Rube ´n E. Loron ˜o, Oasis, Calle 12A+7, Cholul, Me ´rida,
Yucata ´n, Me ´xico, E-mail: email@example.com. Arturo Bates,
Centro Me ´dico Veterinario del Oriente, Tizimı ´n, Yucata ´n, Me ´xico,
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