Am. J. Trop. Med. Hyg., 75(3), 2006, pp. 461–469
Copyright © 2006 by The American Society of Tropical Medicine and Hygiene
GENETIC RELATIONSHIPS AMONG MAYARO AND UNA VIRUSES SUGGEST
DISTINCT PATTERNS OF TRANSMISSION
ANN M. POWERS,* PATRICIA V. AGUILAR, LAURA J. CHANDLER, AARON C. BRAULT, TIFFANY A. MEAKINS,
DOUGLAS WATTS, KEVIN L. RUSSELL, JAMES OLSON, PEDRO F. C. VASCONCELOS,
AMELIA TRAVASSOS DA ROSA, SCOTT C. WEAVER, AND ROBERT B. TESH.
Centers for Disease Control and Prevention, Division of Vector-Borne Infectious Diseases, Fort Collins, Colorado; Center for
Tropical Diseases, Department of Pathology, and Department of Microbiology and Immunology, University of Texas Medical
Branch, Galveston, Texas; US Naval Medical Research Center Detachment, Lima, Peru; Department of Arbovirology and
Hemorrhagic Fevers, Instituto Evandro Chagas, Belem, Para, Brazil
Abstract. Mayaro and Una viruses (MAYV, UNAV) are mosquito-borne alphaviruses that may cause an acute
febrile illness characterized by headache, retro-orbital pain, and rash that may progress to a severe and prolonged
arthralgia. MAYV was first isolated in Trinidad in 1954, and UNAV was first identified in northern Brazil in 1959. Since
then, numerous isolates of these agents have been made from humans, wild vertebrates, and mosquitoes in several
countries in northern South America. Serological evidence suggests that these viruses are also present in portions of
Central America. Because little is known about the natural transmission cycle of MAYV and virtually nothing is known
about UNAV transmission, 63 isolates covering the known geographic and temporal ranges were used in phylogenetic
analyses to aid in understanding the molecular epidemiology. Approximately 2 kb from the E1 and E2 glycoprotein
genes and the complete 3⬘ non-coding region were sequenced. Phylogenetic analyses of these sequences indicated that
two distinct genotypes of MAYV exist with a distinct clade consisting exclusively of UNAV (previously designated as
a subtype of MAYV). One MAYV genotype (genotype D) contains isolates from Trinidad and the northcentral portion
of South America including Peru, French Guiana, Surinam, Brazil, and Bolivia. All of these isolates are highly conserved
with a nucleotide divergence of < 6%. The second MAYV genotype (genotype L) contains isolates only from Brazil that
are highly conserved (< 4% nucleotide divergence) but are quite distinct (15–19%) from the first genotype isolates.
These analyses provide possible explanations for the natural ecology and transmission of MAYV and UNAV.
complex, are far well less characterized, presumably because
the extent of their ability to cause human illness is poorly
understood.
MAYV was first isolated in 1954 from human sera recovered from febrile patients in Trinidad.6 There have been four
documented epidemics of MAYV disease providing the few
opportunities to study the virus during outbreaks of human
illness.7–11 One outbreak in Belterra, Brazil, during 1978 that
was extensively studied provided the first detailed description
of MAYV epidemiology.6,7 During this outbreak, virus isolates were recovered only from Haemagogus janthinomys
mosquitoes, predominantly in the forest canopy where this
mosquito is most typically found. However, two isolates were
made from this mosquito species at ground level, suggesting
that canopy-dwelling mosquitoes could transmit the virus to
humans.12 During the same study, investigators found antibodies against MAYV in only 1% of the birds captured but
27% of marmoset (Callithrix) sera sampled were positive,
indicating that tree-dwelling primates could be a primary vertebrate host of MAYV. The studies during this outbreak represented the first time a specific insect vector was identified in
the transmission of epidemic MAYV and suggested a sylvatic
vertebrate host may have contributed to amplification during
the outbreak. However, the lack of human cases after the
epidemic and inability to recover virus from Hg. janthinomys
the following year indicated that the maintenance cycle had
not yet been identified. Two additional small outbreaks in
Brazil were recorded in 1991 in Benevides, Pará State, and
Peixe, Tocantins State, in 1991, when two isolates of MAYV
were obtained in each municipality from febrile patients and
several other patients showed specific IgM antibodies. Additionally, 15 MAYV strains were isolated from Hg. janthinomys during the outbreaks (Travassos da Rosa and Vasconcelos, unpublished information); this was the same vector that
was incriminated during previous outbreaks. Additional field
INTRODUCTION
Alphaviruses cause two distinct, general disease patterns:
encephalitis with or without demyelination or febrile illness
with persistent arthralgia. Each alphavirus causes only one of
these disease patterns, but the clinical syndromes are not segregated geographically. For example, the alphaviruses present
in South America are Venezuelan equine encephalitis virus
(VEEV), eastern equine encephalitis virus (EEEV), western
equine encephalitis virus (WEEV), Mayaro virus (MAYV),
Una virus (UNAV), Pixuna virus (PIXV), Aura virus
(AURAV), and Trocara virus (TROCV). The first three, as
indicated by their names, cause encephalitis, MAYV and
UNAV cause febrile illness with an accompanying prolonged
and severe arthritis, whereas PIXV, AURAV, and TROCV
are not known to cause serious human illness.1
In addition to the distinct clinical illness patterns, alphaviruses have several different arthropod-borne transmission
cycles that allow the viruses to be maintained in nature. The
most well characterized of these transmission cycles are those
of the equine encephalitis viruses, VEEV, EEEV, and
WEEV. Enzootic VEEV is typically transmitted between
small rodents and mosquitoes of the subgenus Culex melanoconion.2 WEEV and EEEV are better known as viruses that
are maintained in bird–mosquito transmission cycles. However, South American EEEV strains may be maintained in
mosquito–rodent–mosquito cycles similar to that of VEE
complex viruses.3–5 These viruses have received much attention because of the severity of disease when they infect human or equine populations. Viruses such as MAYV and
UNAV, new world members of the Semliki forest antigenic
* Address correspondence to Ann M. Powers, Centers for Disease
Control and Prevention, Division of Vector-Borne Infectious Diseases, Fort Collins, CO 80522. E-mail: [email protected]
461
462
POWERS AND OTHERS
and laboratory studies identified several mosquitoes that
could serve as potential vectors of MAYV,13,14 as well as
several other species of vertebrates that were found to have
antibodies against MAYV.15–21 Despite these efforts, a maintenance cycle for MAYV has not been clearly elucidated.
UNAV, the closest genetic relative of MAYV, is even less
well understood. Little is known about the epidemiology of
UNAV, and virtually no studies have been undertaken to
identify a transmission cycle for this virus. Periodically, isolates of UNAV have been made from a variety of mosquito
species including Psorophora ferox and Ps. albipes,22–24 but
there is no indication that these are the predominant species
involved in virus maintenance or epidemic transmission. Serosurveys have identified antibodies against UNAV in birds,
horses, and humans25–28; however, the extent of viral distribution and human disease risk is unknown.
Until recently, characterization of MAYV and UNAV has
been performed serologically by investigating plaque characteristics or by comparing their pathogenesis in experimentally
infected animals.20,29–33 While all the strains examined in previous serological studies indicated the MAYV or UNAV under study were the same species, some specific tests such as
plaque reduction/neutralization, complement fixation, or
hemagglutination inhibition assays have shown distinctions
among strains,8,34 suggesting different levels of virulence. Attempts to correlate these minor antigenic differences with
biologic significance have not yet been successful. However,
the observation that some large plaque variants produce more
intense lesions in connective tissue of infant mice35 and that
different strains varied in their pathogenicity for adult mice13
has led to speculation that virulence differences may exist
among virus strains.
Recently, molecular methodologies have become valuable
tools to genetically type viruses and to provide insight into the
nature of viral transmission cycles and epidemiologic patterns.36–42 In this study, we used this approach to analyze > 60
strains of MAYV and UNAV isolated over the past 40 years,
covering the known geographic range of these viruses (Figure
1). Our molecular analyses, combined with serological comparisons, give clearer indications of the genetic relationships
among these viruses and suggest mechanisms for their maintenance in nature.
MATERIALS AND METHODS
Viral growth and RNA extraction. Monolayer cultures of
baby hamster kidney cells (BHK-21) or Vero cells in 25-cm2
flasks were infected at a multiplicity of infection (moi) of 0.05
of each virus listed in Table 1. Infected monolayers were
observed daily until cytopathic effect (CPE) was evident in >
90% of the cells. At this time, a 250-␮L aliquot of supernatant
was mixed with 250 ␮L Trizol-LS (BRL Laboratories, Gaithersburg, MD). From the remaining cell culture supernatant,
virus was concentrated by precipitation with polyethylene glycol and RNA extracted from the viral pellet using Trizol as
previously described.43,44 Trizol slurries were stored at −80°C
until RNA was extracted.
Trizol samples were mixed with 2 ␮L of yeast tRNA
(Sigma, St. Louis, MO) and 200 ␮L of chloroform. After vortexing, the samples were centrifuged to separate the phases.
The aqueous phase was mixed with isopropanol to precipitate
the RNA, which was subsequently collected by centrifuga-
FIGURE 1. Distribution of MAYV and UNAV strains. Symbols
indicate regions where isolates of each virus have been obtained.
tion. The RNA pellet was resuspended in 20 ␮L of RNasefree water containing RNase inhibitor (Promega, Madison,
WI) to prevent degradation until subsequent reverse transcription.
Reverse transcription and amplification. A 5-␮l aliquot of
the extracted RNA was mixed in a buffered solution with
dNTPs, RNase inhibitor, dithiothreitol, and T25-C or V
primer designed to anneal to the poly-adenylation sequence.
The mixture was placed in a 42°C bath for 2 minutes before
200 U of Superscript II (Promega) were added and cDNA
was synthesis allowed to proceed for up to 12 hours.45 The
resulting cDNA was amplified by polymerase chain reaction
(PCR) using Pfu Turbo Polymerase (Stratagene, La Jolla,
CA) and primers (40 pmol) designed to amplify a portion of
E2, E1, and the 3⬘ non-coding region (NCR) from 9368
through the end of the genome (Table 2; Figure 2). Amplicons were either cleaned and sequenced directly or subcloned
and sequenced from the plasmids.
Sequencing and phylogenetic analysis. Sequencing reactions were performed on the PCR amplicons using Big Dye
3.0 on an ABI 377 automated sequencer or the DTCS Quick
Start Kit on a Beckman capillary sequencer (see Table 2 for
sequencing primers used). Sequences were aligned using the
GAP and PILEUP program in the Genetics Computer Group
package46 with manual refinements to maintain codon homology. Phylogenetic analyses were performed using maximum
parsimony (heuristic algorithm), neighbor-joining distance
matrix algorithms (Kimura 3 parameter and F84 corrections),
and maximum-likelihood approaches (Quartet puzzling with
the HKY85 least squares variant) within the PAUP program.47 Both nucleotide sequence covering the entire amplicon and amino acid sequence from the E2/E1 coding region
were used in the analyses. Each clade was also resampled
individually by maximum likelihood to increase resolution
among highly conserved sequences. Other members of the
463
GENETIC RELATIONSHIPS AMONG MAYARO AND UNA VIRUSES
TABLE 1
Summary of Mayaro and Una viral isolates examined
Strain
Location state/department, country
Isolation date
Passage history
Source
Accession no.
Mayaro viruses
07-18066-99
ARV 0565
BeAn 343102
BeAr 30853
BeAr 350396
BeAr505411
BeH 186258
BeH 256
BeH 342912
BeH 343148
BeH 343155
BeH 343178
BeH 394881
BeH 407
BeH 473130
BeH 428890
BeH 504378
BeH 504639
BeH 506151
D218
DEF533
FSB279
FSB309
FSB311
FSB319
FSB323
FSC497
FSC498
Guyane
IQD2668
IQD4881
IQD5316
IQD5364
IQT 2849
IQT 4235 (CH)
IQU 2939
IQU 2950
IQU 3056
IQU 3132
MFI0231
Obs 2209
Obs 2248
Obs 2251
Obs 2340
Obs 6161
Obs 6443
Obs 6515
Obt2191
Ohio (#96-104)
TRVL 4675
TRVL 15537
Uruma
Huanuco, Peru
San Martin, Peru
Para, Brazil
Para, Brazil
Para, Brazil
Para, Brazil
Amapa, Brazil
Para, Brazil
Para, Brazil
Para, Brazil
Para, Brazil
Para, Brazil
Para, Brazil
Para, Brazil
Para, Brazil
Para, Brazil
Para, Brazil
Goias, Brazil
Tocantins, Brazil
Surinam
Loreto, Peru
Bolivia
Bolivia
Bolivia
Bolivia
Bolivia
Cuzco, Peru
Cuzco, Peru
French Guiana
Loreto, Peru
Loreto, Peru
Loreto, Peru
Loreto, Peru
Loreto, Peru
Loreto, Peru
Loreto, Peru
Loreto, Peru
Loreto, Peru
Loreto, Peru
Iquitos, Peru
Tumbes, Peru
Huanuco, Peru
Huanuco, Peru
Ayacucho, Peru
Ucayali, Peru
Ucayali, Peru
Cuzco, Peru
Loreto, Peru
Loreto, Peru
Mayaro County, Trinidad
Rio Grande Forest, Trinidad
Uruma, Bolivia
July, 1999
April, 1995
May, 1978
May, 1961
August, 1978
March, 1991
June, 1970
April, 1955
April, 1978
April, 1978
April, 1978
April, 1978
June, 1981
1955
May, 1988
December, 1984
1991
1991
1991
September, 1984
March, 2003
February, 2002
March, 2002
March, 2002
April, 2002
April, 2002
March, 2003
March, 2003
1996
May, 2002
February, 2003
March, 2003
February, 2003
February, 1996
August, 1997
2000
June, 2000
June, 2000
2000
September, 2002
March, 1995
May, 1995
May, 1995
June, 1995
January, 1998
February, 1998
1998
April, 2002
1995
August, 1954
March, 1957
March, 1955
V-1, BHK-1
SM-1, V-1
Sm-4, V-1
SM-1, V-1
V-1
SM-1, V-1, BHK-1
V-1
SM-8, V-1
SM-2, V-1
V-1
V-1
V-1
SM-2, V-1
P-?, V-2
V-1
SM-1, V-1
SM-1, V-1
SM-1, V-1
SM-1, V-1
P-1, C6/36-2, BHK-1
Human serum
Human
Monkey
Ixodes spp.
Haemagogus spp.
Haemagogus janthinomys
Human
Human serum
Human
Human
Human
Human
Human
Human
Human
Human
Human
Human
Human
Human serum
Human serum
Human serum
Human serum
Human serum
Human serum
Human serum
Human serum
Human serum
Human serum
Human serum
Human serum
Human serum
Human
Human
Human
Human
Human
Human
Human serum
Human
Human serum
Human
Human
Human
Human
Human
Human serum
Human
Human serum
Mansonia venezuelensis
Human
DQ487401
DQ487397
DQ487389
DQ487378
DQ487388
DQ487382
DQ487408
DQ487381
DQ487387
DQ487413
DQ487390
DQ487391
DQ487414
DQ487380
DQ487379
DQ487383
DQ487407
DQ487392
DQ487386
DQ487409
DQ487425
DQ487424
DQ487423
DQ487422
DQ487421
DQ487420
DQ487419
DQ487418
DQ487385
DQ487429
DQ487428
DQ487427
DQ487426
DQ487403
DQ487410
DQ487415
DQ487394
DQ487402
DQ487416
DQ487430
DQ487399
DQ487398
DQ487396
DQ487406
DQ487393
DQ487400
DQ487404
DQ487431
DQ487405
DQ487369
DQ487384
DQ487395
Una viruses
788382
BeAr 13136
BeAr 379631
BeAr 416216
CoAn 20-26-70
BT 1495-3
CbaAn 979
CoAr 2518
MAC 150
PE 10800
ZPC 195
Trinidad
Para, Brazil
Goias, Brazil
Roraima, Brazil
Colombia
Bocas del Toro, Panama
Cordoba, Argentina
Valle, Colombia
Miranda, Venezuela
Loreto, Peru
Rio Claro, Venezuela
1978
September, 1959
1980
1983
1972
1960–1961
January, 1964
April, 1964
October, 1997
November, 1996
August, 1998
P?, SM3, BHK-1
SM-6, BHK-1
P-?, V-1
SM-1, V-1
SM-9, BHK-1
P-?, SM-8, BHK-1
SM-9, BHK-1
SM-3, BHK-1
V-1
V-3, BHK-1
V-1, BHK-1
Psorophora spp.
Psorophora ferrox
Psorophora ferox
Aedes serratus
Dasyprocta fuliginosa
Mosquitoes
Horse
Psorophora ferrox
Hamster
Psorophora ferrox
Hamster
DQ487417
DQ487373
DQ487412
DQ487411
DQ487370
DQ487376
DQ487374
DQ487377
DQ487375
DQ487371
DQ487372
V-2
V-2
V-2
V-2
V-2
V-2
V-2
p-?, V-1
C6/36-1
C6/36-1
C6/36-1
V-2
C6/36-2, V-1
C6/36-2, V-1
C6/36-1, V-1
C6/36-1, V-1
V-1, BHK-1
C6/36-1, V-1
C6/36-1
V-1, BHK-1
C6/36-2, BHK-1
V-1, BHK-1
SM-1, V-1
C6/36-2, V-1, BHK-1
C6/36-1, V-1, BHK-1
C6/36-1, V-2, BHK-1
V-2
p-?, V-1
SM-13, V-1
SM-5, V-1, BHK-1
P-?
V, Vero cells; BHK, baby hamster kidney cells; C6/36, Aedes albopictus mosquito cells; SM, suckling mouse; P, passage of undocumented cell or host type; ?, unknown number of passages.
464
POWERS AND OTHERS
TABLE 2
Polymerase chain reaction amplification and sequencing primers used
for MAY and/or UNA viruses
Name
Sequence
PCR primers
9368(+)
ACCAGTGGTGCAACCATAAACC
9397(+)
GCARWGCRCATGGMTGGCC
10344(−)
TCGTGTTTRCACACGTCAGC
10475(+)
10051(+)
9400(+)
10247A
CACGTTCCATACACGCAGACTCC
GGAGCATAYTGCTTCTGCGACAC
GGCAATGCGCACGGATGGC
TACCCCNTTYATGTGGGG
10247B
CCCCACATRAANGGGTA
T25C
CTTTTTTTTTTTTTTTTTTTTTTTTT
T215V
VTTTTTTTTTTTTTTTTTTTTTTTTT
Sequencing primers
9368(+)
ACCAGTGGTGCAACCATAAACC
9397(+)
GCARWGCRCATGGMTGGCC
9597(−)
10344(−)
CCGATAGTAACAGGGACAAC
TCGTGTTTRCACACGTCAGC
10475(+)
9899(+)
10051(+)
10379(+)
10734(+)
10130(+)
10576(+)
10576(−)
11006(+)
9400(+)
11006(−)
10247A
CACGTTCCATACACGCAGACTCC
CAAATGCAGGTGGTGGAGAC
GGAGCATAYTGCTTCTGCGACAC
CTGGTAGGTTTGGAGACATTC
GTGCACTGTATCTACATGCAC
CCAWCRCTYAACCTGGAG
GTTAGGGCCATGAAYTGTGCGG
CCGCACARTTCATGGCCCTAAC
GGGTTCARCGACTGGCAGG
GGCAATGCGCACGGATGGC
CCTGCCAGTCGYTGAACCC
TACCCCNTTYATGTGGGG
10247B
CCCCACATRAANGGGTA
MAY or
UNA specific
UNA and
MAY
UNA and
MAY
UNA and
MAY
UNA
UNA
UNA
UNA and
MAY
UNA and
MAY
UNA and
MAY
UNA and
MAY
UNA and
MAY
UNA and
MAY
MAY
UNA and
MAY
UNA
UNA
UNA
UNA
MAY
MAY
UNA
UNA
UNA
UNA
UNA
UNA and
MAY
UNA and
MAY
alphavirus genus were included as outgroup members. Bootstrap resampling (1,000 replicates) provided estimates of confidence on the groups generated in each analysis.48
Production of immune sera. BALB/C mice were used to
generate immune sera to five diverse strains of UNAV
(Tables 3 and 4). Animals received a single intraperitoneal
injection of virus, and ∼4 weeks after inoculation, mice were
injected intraperitoneally with sarcoma 180 cells to produce hyperimmune ascitic fluid. Abdominal ascitic fluid was
removed between 1 and 2 weeks after injection of the sarcoma cells and was used in serological analyses as described
below.
Serological analyses. Complement fixation (CF) and hemagglutination inhibition (HI) assays were performed on selected UNAV isolates to further characterize the relationships identified with genetic analyses. Two-way tests were
completed with all five sera and antigens were examined. CF
tests were performed using a microtechnique49 with two full
units of guinea pig complement. Titers were recorded as the
highest dilutions giving 3+ or 4+ fixation of complement on a
scale of 0 to 4+. HI tests were also performed using a microtechnique, as described previously.49 Immune sera and ascitic
fluids for the HI tests were acetone-extracted, and antigens
were tested at pH 6.2.
RESULTS
Sequence analysis. The sequence of nucleotides covering
the 3⬘ portion of the E2 protein, the entire E1 protein, and the
3⬘ non-coding region (NCR) was determined for each MAYV
and UNAV listed in Table 1; these strains were collected
from a broad geographic range as depicted in Figure 1. This
amplicon is a region that has previously been shown to contain phylogenetically informative characters for many alphaviruses36; therefore, the PCR products were analyzed by
comparison of the nucleotide and deduced amino acid sequences with those of other previously sequenced alphaviruses. As expected, pairwise comparisons and phylogenetic
analyses showed the isolates to be most closely related to
other members within the Semliki Forest antigenic complex.
Both nucleotide and E2/E1 amino acid sequences were used
in the analyses; however, because of the extremely high degree of nucleotide conservation among the MAYV strains,
amino acid analyses were less informative and generally produced topologies with less resolution. All analyses produced
the same three distinct genotypes, but there were analysisspecific variations among the strains within a given genotype.
For example, the high degree of genetic conservation frequently yielded several hundred equally parsimonious topologies at the terminal branches.
In all analyses performed, all MAYV strains formed a
monophyletic group. Pairwise comparisons showed the
MAYV strains to range from 0.05% to 18.2% nucleotide sequence divergence. These MAYV strains segregated into two
distinct genotypic clades, each of which consisted of members
with very little genetic divergence (Figure 3). The first clade
(genotype D) contained isolates from 1954 to 2003, covering
a geographic range spanning South America that included
Trinidad, Brazil, French Guiana, Surinam, Peru, and Bolivia.
This clade also contained the strain Uruma, which was reported as a new human pathogen in 1959 but never previously
published as a MAYV strain.50 The most closely related virus
strains within clade D were only 0.05% divergent with the
maximum nucleotide diversity reaching 5.9%. The second
clade (genotype L) contained only six isolates; all were geographically limited to the northcentral region of Brazil and
were genetically similar (ranging from 0.1% to 3.0% nucleotide sequence divergence), even though the isolations were
made over a broad temporal range (1955–1991). Members of
genotype D were 15–18% divergent from strains of genotype
L at the nucleotide level. Whereas bootstrap support for clustering within either clade was strong in only two instances
(two early isolates from Trinidad and several recent isolates
from Peru and Bolivia), there was 100% support for the distinction between the two clades.
As with the MAYV, the UNAV strains formed a monophyletic group. However, unlike the MAYV strains that exhibited a high degree of genetic relatedness within clades, the
UNAV isolates were much more genetically diverse. Pairwise
comparisons revealed nucleotide divergence levels between
2.5% and 27.7% with no apparent clustering based on geog-
465
GENETIC RELATIONSHIPS AMONG MAYARO AND UNA VIRUSES
FIGURE 2.
MAYV genome organization and amplicon region.
raphy or time of isolation. However, the limited number of
isolates may contribute to the lack of any discernible patterns.
Antigenic analyses. To further evaluate the relationships of
the UNAV in this study, several strains were analyzed for
antigenic differences of biologic significance. Antibodies to
five strains were generated in mice and used in HI and CF
assays to determine antigenic relationships against homologous and heterologous viruses (Tables 3 and 4, respectively).
In general, the homologous virus titers were the highest in
both testing formats; however, several exceptions to this trend
were noted. For example, when ZPC195 strain (from Venezuela) was used as antigen, antibody titers from sera of
BeAr13136 (Brazil) and CbaAn979 (Argentina) infected
mice were up to 4-fold higher than the homologous titers in
HI and/or CF assays, suggesting that this antigen contained
broadly conserved epitopes. Curiously, Peruvian strain
PE10800 (the virus most closely related to ZPC195 in the
serological analysis) always had lower titers against ZPC195
antigen or serum than more phylogenetically distant strains.
In comparisons of all strains included in this analysis, there
was no instance where there were 4-fold or greater differences between strains in both directions. Whereas the oneway 4-fold differences did occasionally exist in both test formats, this is only sufficient to classify strains as antigenic subtypes.51 The UNAV strains from Peru and Colombia were
indistinguishable by CF and HI assays with the strain from
Venezuela, which is the most closely related antigenically to
the Peruvian/Colombian pair. The strains from Brazil and
Argentina were closely related to each other but more distant
from the remaining viruses. These antigenic subtype distinctions demonstrated general geographic alignments as well as
an agreement with the phylogenetic analyses performed in
this study.
TABLE 3
Results of hemagglutination inhibition antibody tests showing the
relationships between selected UNAV isolates
DISCUSSION
Mayaro viruses are one group of alphaviruses that are of
resurging interest in South America. Recent reports suggest
that changing demographics and land use practices could alter
the frequency of human illness caused by MAYV.8,9,52 The
most detailed studies of the virus occurred during an outbreak
near Belterra, Brazil, in 1978. Through the epidemiologic and
entomologic studies, a description of how a MAYV epidemic
spreads and amplifies was proposed.7,8,12 Unfortunately, no
clear understanding of how the virus is maintained afterward
or what initiates an epidemic has been recorded. To accomplish this, extensive field studies to monitor human illness,
non-human seroprevalence, and mosquito infection rates
would be ideal. However, in the absence of such longitudinal
field studies, molecular genetic analysis of existing MAYV
isolates can provide clues as to its maintenance patterns and
geographic distribution; this approach may also help to more
effectively design field studies. We examined > 60 isolates of
MAYV in an attempt to ascertain relationships among them.
We also genetically analyzed the most closely related virus,
UNAV, to determine if the patterns of maintenance for arthritis-causing alphaviruses in South America are consistent.
Our genetic analyses showed that the MAYV strains are
monophyletic and form two very distinct genotypic lineages.
Interestingly, the two genotypes are sympatric in the Amazon
basin of Brazil, but they are > 15% divergent at the nucleic
acid level. There is no evidence of alternate genetic types or
other clades of MAYV in this same geographic area even
though there are isolates from both the widely dispersed (D)
and limited (L) genotypes from approximately the same year.
Furthermore, even with isolates spanning over 40 years, there
were no isolates identified that were distinct from either the L
or D genotypes. This information suggests that distinct transTABLE 4
Results of complement fixation titers comparing selected UNAV strains
Antigens* (4 units)
Antigens*
UNAV antisera
Be Ar 13136
Cba An 979
PE 10800
Co Ar 2380
ZPC 195
UNAV antisera
Be Ar 13136
Cba An 979
PE 10800
Co Ar 2380
ZPC 195
Be Ar 13136
Cba An 979
PE 10800
Co Ar 2380
ZPC 195
2,560
2,560
320
320
320
1,280
2,560
640
640
640
2,560
1,280
1,280
1,280
640
2,560
2,560
1,280
1,280
640
2,560
1,280
1,280
1,280
1,280
Be Ar 13136
Cba An 979
PE 10800
Co Ar 2380
ZPC 195
1,024
512
64
128
128
512
1,024
32
32
128
512
512
128
128
128
512
256
128
128
256
512
512
64
64
128
* Bold indicates homologous titers.
* Bold indicates homologous titers.
466
POWERS AND OTHERS
FIGURE 3. Phylogram of genetic relationships among the MAVY and UNAV strains sequenced in this study. Numbers are bootstrap support
values for the specific clades.
mission patterns may have played a role in the evolution and
current maintenance of MAYV.
The very close genetic relatedness of isolates within a given
genotype is suggestive of a transmission mechanism that continually circulates viruses and is unimpeded by significant barriers to dispersal that would lead to divergent evolution. An
example of another alphavirus that exhibits this molecular
epidemiologic pattern is North American EEEV. EEEV, in
North America, is maintained in a very well-characterized
enzootic cycle involving Culiseta melanura mosquitoes and
avian vertebrate hosts.53,54 Molecular phylogenetic studies of
EEEV have shown that North American strains have > 98%
nucleotide identity and form a single, highly conserved lineage grouped to some extent by geography and year of isolation.55–57 A virtually identical pattern is observed within
each clade of MAYV in this study, suggesting that the maintenance may involve bird hosts that broadly disperse the viruses maintaining genetic homogeneity over extensive periods of time. As with the North American EEEV strains, some
temporal groupings in the terminal branches of MAYV were
observed and may reflect regional or local foci during a 2- to
3-year period. One example was the cluster of 13 strains from
Peru and Bolivia collected in 2002–2003. This clustering may
only have been identified because numerous isolates were
obtained from the same area during a short time frame allowing only this degree of resolution in the genetic analyses.
Similar localized patterns of virus transmission have been
documented for St. Louis encephalitis virus58 and West Nile
virus,59 which are maintained in mosquito–bird transmission
cycles. While there is little information regarding the seroprevalence of MAYV in birds, there have been isolations
from avians and some evidence that birds may be involved in
the enzootic cycles.15,52
More intriguing than the low degree of genetic diversity
within clades is the distinct and complete divergence of the
two genotypes of MAYV with no other apparent genetic
types existing. This is particularly curious because of the overlapping geography and the fact that both genotypes contain
isolates spanning 30 or more years. This would suggest that
both lineages are maintained independently in enzootic main-
GENETIC RELATIONSHIPS AMONG MAYARO AND UNA VIRUSES
tenance cycles in discrete foci leading to divergent gene flow
and that neither has gone extinct. In this scenario, transmission by resident vertebrates (in genotype L), rather than
widely dispersing avian hosts, would seem likely. Detection of
antibodies against MAYV in sloths, marmosets, other primates, rodents, and numerous other non-flying mammals12,16,19,60,61 provides evidence that focal enzootic transmission of the virus may indeed be a component of the transmission cycle that led to the evolution of two distinct MAYV
genotypes.
Because several MAYV isolates have been obtained from
Haemagogus sp. mosquitoes, it has also been postulated that
MAYV is maintained in constantly moving waves or “wandering epizootics” by non-human primates similar to the pattern that has been described for the sylvan cycle of yellow
fever virus (YFV).52 The fact that distinct genotypes exist
would suggest that, at most, the wandering population hypothesis is only partially applicable. As with MAYV, this
finding of discrete genetic groups of YFV in the Amazon
region has also been used to argue against the concept of
continuous movement of the virus within primate populations.40 Additionally, if a single, intermixed population of
MAYV was continually moving across the broad geographic
range of tropical South America, a continual evolution of the
virus from a hypothetical ancestor would be expected. Our
genetic analysis showed no evidence of this but rather a true
conservation of the genome over time and space.
In contrast to the high degree of genetic homogeneity
among the MAYV strains, the UNAV isolates examined in
this study have a very different phylogenetic pattern. The
UNAV strains are monophyletic but have a maximum genetic
diversity of 28%. They are also distinct from the MAYV
strains, with only ∼45% nucleotide identity between the two
species. While some of the UNAV strains isolated by as much
as 35 years apart were quite similar (pairwise comparisons of
as little as 2.5%), others separated by only a few years varied
genetically by 20%. The genetic diversity pattern of UNAV
would suggest that these viruses moved to new ecological
niches and subsequently established discrete enzootic foci in
which the viruses are being maintained, following divergent
evolutionary paths. This would be consistent with transmission by rodents or other vertebrates with limited movement
patterns. Our serological analyses were a further attempt to
ascertain the relationships among the UNA viruses and were
in general agreement with the phylogenetic analyses. Unfortunately, the degree of cross-reactivity among the antisera
produced in our study was too great to distinguish these viruses as more than variants or subtypes. With the paucity of
seroprevalence data for UNAV, the molecular evidence implicating discrete transmission foci provides the most reasonable estimate of enzootic maintenance for the virus at this
time. Of further note for both the UNAV and MAYV strains
is that, whereas the serological analyses indicate that there are
indeed some distinctions, in the absence of biologic characterization of these isolates, it is difficult to assess whether
these subtypes have pathogenic or epidemiologic relevance. It
is also important to emphasize that, whereas MAYV is a
well-known human pathogen, UNAV has not yet been recovered from humans. Further studies are warranted to evaluate
the extent of distribution of MAYV and UNAV and the risk
to human and animal populations in the affected areas. Hopefully, information obtained from the molecular epidemiologic
467
studies presented here will provide information to make more
informed decisions regarding the most appropriate field materials to be collected.
Received October 17, 2005. Accepted for publication April 28, 2006.
Acknowledgments: The authors thank the Centers for Disease Control and Prevention Arbovirus Reference Collection, the UTMB
World Reference Center for Emerging Viruses and Arboviruses, and
Dr. Tad Kochel of the U.S. Naval Medical Research Center Detachment, Lima, Peru for providing the viruses used in this study.
Financial support: A.M.P. was supported in part by NIH Grant AI07536. A.C.B. and P.A. were supported by the James W. McLaughlin
Fellowship Fund and NIH Grant AI-107526. This research was supported in part by Grant AI049725 from the NSF/NIH program on the
ecology of infectious disease, NIH Contract N01-AI30027, and CNPq
grant process 302770/2002-0. This work was also supported by funded
work unit No. 847705 82000 25GB B0016.
Disclaimer: The views expressed in this article are those of the author
and do not necessarily reflect the official policy or position of the
Department of the Navy, Department of Defense, nor the U.S. Government. No official support or endorsement by the Centers for Disease Control and Prevention, Department of Health and Human
Services is intended, nor should be inferred.
Human use statement: Some of the Mayaro isolates were obtained
under the study protocol which was approved by the Naval Medical
Research Center Institutional Review Board (Protocol
# NMRCD.2000.0006) in compliance with all applicable Federal
regulations governing the protection of human subjects.
Authors’ addresses: Ann M. Powers, Aaron C. Brault, and Tiffany
Meakins, Centers for Disease Control and Prevention, Division of
Vector-Borne Infectious Diseases, Fort Collins, CO 80522. Patricia
Aguilar, Laura J. Chandler, Amelia Travassos Da Rosa, Scott C.
Weaver, and Robert B. Tesh, Center for Tropical Diseases, Department of Pathology, and Department of Microbiology and Immunology, University of Texas Medical Branch, Galveston, TX 77555.
Douglas Watts, Kevin Russell, and James Olson, US Naval Medical
Research Center Detachment, Lima, Peru. Pedro Vasconcelos, Department of Arbovirology and Hemorrhagic Fevers, Instituto Evandro Chagas, Belem, Para, Brazil.
Reprint requests: Ann M. Powers, Division of Vector-Borne Infectious Diseases, Centers for Disease Control and Prevention, PO Box
2087, Fort Collins, CO 80522.
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