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Genomic and phylogenetic characterization of
viruses included in the Manzanilla and Oropouche
species complexes of the genus Orthobunyavirus,
family Bunyaviridae
Jason T. Ladner,
1
3Nazir Savji,
2
34 Loreen Lofts,
1
Amelia Travassos da Rosa,
3
Michael R. Wiley,
1
Marie C. Gestole,
1
Gail E. Rosen,
2
Hilda Guzman,
3
Pedro F. C. Vasconcelos,
4
Marcio R. T. Nunes,
5
Tadeusz J. Kochel,
6
1W. Ian Lipkin,
2
Robert B. Tesh
3
and Gustavo Palacios
1
Correspondence
Gustavo Palacios
gustavo.f.palacios.ctr@us.army.mil
Received 5 November 2013
Accepted 17 February 2014
1
Center for Genomic Sciences, United States Army Medical Institute for Infectious Disease,
Frederick, MD, USA
2
Center for Infection and Immunity, Mailman School of Public Health, Columbia University,
New York, NY, USA
3
Center for Biodefense and Emerging Infectious Diseases, Department of Pathology,
University of Texas Medical Branch, Galveston, TX, USA
4
Department of Arbovirology and Hemorrhagic Fevers, Instituto Evandro Chagas,
Ananindeua, Brazil
5
Center for Technological Innovation, Instituto Evandro Chagas, Ananindeua, Brazil
6
Virology Department, Naval Medical Research Unit Six, Lima, Peru
A thorough characterization of the genetic diversity of viruses present in vector and vertebrate
host populations is essential for the early detection of and response to emerging pathogenic
viruses, yet genetic characterization of many important viral groups remains incomplete. The
Simbu serogroup of the genus Orthobunyavirus, family Bunyaviridae, is an example. The Simbu
serogroup currently consists of a highly diverse group of related arboviruses that infect both
humans and economically important livestock species. Here, we report complete genome
sequences for 11 viruses within this group, with a focus on the large and poorly characterized
Manzanilla and Oropouche species complexes. Phylogenetic and pairwise divergence analyses
indicated the presence of high levels of genetic diversity within these two species complexes, on a
par with that seen among the five other species complexes in the Simbu serogroup. Based on
previously reported divergence thresholds between species, the data suggested that these two
complexes should actually be divided into at least five species. Together these five species
formed a distinct phylogenetic clade apart from the rest of the Simbu serogroup. Pairwise
sequence divergences among viruses of this clade and viruses in other Simbu serogroup species
complexes were similar to levels of divergence among the other orthobunyavirus serogroups. The
genetic data also suggested relatively high levels of natural reassortment, with three potential
reassortment events present, including two well-supported events involving viruses known to
infect humans.
3These authors contributed equally to this paper.
4Present address: School of Medicine, New York University, New York, NY, USA.
1Present address: Viral and Rickettsial Diseases Department, Naval Medical Research Center, Silver Spring, MD, USA.
The GenBank/EMBL/DDBJ accession numbers for the virus isolates sequenced in this paper are JQ675598–JQ675603 and KF967136–KF697162,
full details of which are given in Table 1.
Six supplementary figures and three supplementary tables are available with the online version of this paper.
Journal of General Virology (2014), 95, 1055–1066 DOI 10.1099/vir.0.061309-0
061309 1055
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INTRODUCTION
Globalization of travel and trade, climate change and ever-
growing human population sizes are all contributing to an
increase in the emergence of pathogenic viruses (Lipkin,
2013). Many of these viruses are coming from well-
characterized and expected groups (e.g. influenza, flavi-
viruses), whereas others belong to groups that have largely
been ignored by past surveillance programmes. An example
of the latter are members of the genus Phlebovirus,
which, with the notable exceptions of Rift Valley fever
and Toscana viruses, were generally thought to be of little
current public health importance until the recent emer-
gence of severe fever with thrombocytopenia syndrome
virus (SFTSV) and Heartland virus (HRTV) (McMullan
et al., 2012; Yu et al., 2011). Another, even larger, neglected
group is the orthobunyaviruses. Due to their abundance
and diversity, many orthobunyaviruses have yet to be fully
sequenced and there are probably many others that have
yet to be detected. However, a thorough understanding of
the sequence diversity of such circulating viruses is a
critical part of surveillance and preparedness for future
disease outbreaks.
The genus Orthobunyavirus is the largest in the family
Bunyaviridae with over 170 named viruses corresponding
to 18 different serogroups and 48 species complexes (Elliott
& Blakqori, 2011; Nichol et al., 2005). Although the term
‘serogroup’ is not currently utilized by the International
Committee on Taxonomy of Viruses (ICTV), the concept
of serogroups has played an important historical role in
viral taxonomy (Calisher & Karabatsos, 1988); the classifi-
cation of arthropod-borne viruses (arboviruses) was initially
based on antigenic relationships revealed by serological tests
(Casals, 1957). In general, genetic-based classifications are
starting to supplant antigenic classifications; however, due
to the lack of genetic information for many named viruses in
the family Bunyaviridae, most current taxonomic assign-
ments are still based on serological criteria (Nichol et al.,
2005; Plyusnin et al., 2012). Thus, in this report, in the
interests of continuity and clarity, the term ‘serogroup’ will
continue to be used for a group of serologically related
viruses and the term ‘species complex’ will be used for
ICTV-defined (Nichol et al., 2005) groups of closely related,
differently named viruses whose exact taxonomic status
remains uncertain because of slight antigenic variation or
differences in host range, vector species, geographical
distribution and/or pathogenic potential from the desig-
nated type species. The purpose of the present report was to
explore genetic diversity within the Simbu serogroup of the
genus Orthobunyavirus, a diverse and geographically wide-
spread group that includes important human and livestock
pathogens (Kinney & Calisher, 1981; Saeed et al., 2001a).
The Simbu serogroup currently comprises 22 officially
recognized viruses that have been grouped into seven differ-
ent species complexes (Akabane, Manzanilla, Oropouche,
Sathuperi, Simbu, Shamonda and Shuni; Nichol et al., 2005),
as well as several other recently described viruses that have yet
to be officially assigned to a species (Aguilar et al., 2011;
Figueiredo & Da Rosa, 1988; Goller et al., 2012; Plyusnin et al.,
2012; Saeed et al., 2001b). Full genomes have recently been
obtained for 12 viruses within the Simbu serogroup (Goller
et al., 2012); however, these genome sequences are not equally
distributed among the seven species complexes. For example,
only one representative, Oropouche virus (OROV), has been
fully sequenced from the Oropouche species complex, and no
complete sequences are available from the Manzanilla species
complex (Kinney & Calisher, 1981). Yet, these are two of the
largest species complexes within the Simbu serogroup, and
the Oropouche species complex is the only one with members
that are known to cause human disease. The lack of complete
genomes for all members of these species complexes impacts
diagnostic capacity (e.g. the ability to identify conserved/
divergent regions for primer design in PCR applications and
for recognition with sequence-based diagnostic methods); it
also prevents the recognition of reassortants.
Here, we utilized high-throughput sequencing technologies
to improve our understanding of the diversity and
evolutionary history of these two species complexes
by obtaining full genome sequences for 11 previously
uncharacterized viruses, including the three remaining
members of the Oropouche species complex, four of the
five members of the Manzanilla species complex and four
other unassigned viruses that have demonstrated genetic
and/or antigenic similarities to one of these two species
complexes. In order to compare the sequences with
previous taxonomic characterizations, serological compari-
sons were conducted among these 11 uncharacterized
viruses and the other fully sequenced members of these two
species complexes.
RESULTS AND DISCUSSION
Genome sequences
Genome sequences for 11 viruses (Table 1) were obtained
through de novo assembly from either 454 (Roche) or
Illumina sequences. Each sequenced orthobunyavirus
genome consisted of three distinct RNA segments, and
the sizes and organization of the ORFs were generally
consistent with what has been described previously for the
genus (Plyusnin et al., 2012) (Table S1, available in the
online Supplementary Material). The 39terminal sequence
was obtained for 18 segments (nine different viruses) and
the 59terminal sequence was obtained for seven segments
(five different viruses). In all cases, the 10 most terminal
nucleotides were identical to those that have been reported
previously for the genus (Plyusnin et al., 2012). Segments
without sufficient coverage to assemble the ends were
completed using primers targeting these conserved ter-
minal sequences. The large (L) genome segment of
members of the genus Orthobunyavirus contains a single
ORF that encodes an RNA polymerase; in our sequences,
this ORF ranged in size from 6756 to 6783 bases. The
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medium-sized (M) segment also contains a single ORF,
which encodes a polyprotein that is co-translationally
cleaved into two envelope glycoproteins and a non-
structural protein. The M-segment ORF of the 11 viruses
ranged in size from 4254 to 4299 bases. The smallest (S)
segment typically contains two overlapping ORFs, which
code for a nucleocapsid protein (NP) and a non-structural
(NSs) protein. The NP ORF of the 11 viruses ranged in size
from 693 to 699 bases, whilst the NSs protein ORF ranged
from 273 to 288 bases, with two exceptions: Utinga (UTIV)
and Utive (UVV) both contained severely truncated,
presumably non-functional, versions of the NSs protein
ORF due to multiple nonsense mutations (81 bases, 27 aa).
When functional, the NSs proteins of orthobunyaviruses
have been shown to act as IFN antagonists (Elliott & Weber,
2009), and therefore it is reasonable to suggest that loss of
the NSs protein in UTIV and UVV may have altered the
virulence of these viruses. Similar loss of the NSs protein has
been reported in several other orthobunyavirus serogroups,
including Anopheles A and B, Tete and Wyeomyia
(Chowdhary et al., 2012; Mohamed et al., 2009); however,
to our knowledge, this is the first description of a single
serogroup with both members that contain and members
that lack a NSs protein.
Genus-level phylogenetic relationships
Phylogenetic analyses of the three genome segments
confirmed that the 11 sequenced viruses were all genetically
closely related to the previously sequenced Simbu sero-
group viruses. The Simbu serogroup formed a monophyletic
clade in both the M- and S-segment trees (bootstraps566.9
and 100, respectively; Figs S1 and S2), and in the L-segment
tree the serogroup was paraphyletic, also including Leanyer
virus (LEAV; Fig. 1) (Savji et al., 2011). Uncertainty in
placing LEAV was the main reason for a low bootstrap in the
M-segment tree; without LEAV, the Simbu serogroup clade
was supported in 90 % of bootstrap analyses. Our extended
genetic sampling of the Oropouche and Manzanilla species
complexes illuminated a deep evolutionary divide within the
Simbu serogroup; one phylogenetic clade (clade A) included
the Manzanilla and Oropouche species complexes and a
second clade (clade B) included the other five Simbu species
complexes (Figs 1, S1 and S2) (Kinney & Calisher, 1981).
These monophyletic clades were well supported across all
three genomic segments with bootstrap support ranging
from 97.5 to 100 %, and there was no evidence of
reassortment between these clades.
In 1981, Kinney and Calisher used a combination of
serological analyses to provide finer levels of classification
within the Simbu serogroup (Kinney & Calisher, 1981); the
two genetic clades seen in our analysis are consistent with
the serocomplexes they identified. Clade B corresponds
completely to their original Simbu serocomplex, whereas
the Oropouche and Manzanilla species complexes (clade
A) each correspond to unique serocomplexes. Pairwise
genetic similarities at the amino acid level between clades A
Table 1. Virus isolates sequenced in this study
Virus Strain Source Locality Year Species* GenBank accession nos Reference
MANV TRVL 3587 Alouatta seniculus (red howler monkey) Trinidad 1954 Manzanilla KF697148–KF697150 Anderson et al. (1960)
INGV SA An 4165 Hyphanturgus ocularis (spectacled weaver) South Africa 1959 Manzanilla KF697139–KF697141 McIntosh et al. (1965)
MERV AV 782 Progne subis (purple martin) USA 1964 Manzanilla KF697151–KF697153 Calisher et al. (1969)
Cat Que virusDVN 04-2108 Culex sp. (mosquitoes) Vietnam 2004 Manzanilla JQ675598–JQ675600 Bryant et al. (2005)
BUTV BFS 5002 Culicoides sp. (biting midges) USA 1964 Buttonwillow KF697160–KF697162 Reeves et al. (1970)
FPV Aus Ch 16129 Mosquitoes Australia 1974 Facey’s Paddock KF697136–KF697138 Doherty et al. (1979)
UTIV Be An 84785 Bradypus tridactylus (pale-throated sloth) Brazil 1965 Utinga KF697154–KF697156 Shope et al. (1967)
UVV Pan An 48878 Bradypus variegates (brown-throated sloth) Panama 1975 Utinga KF697157–KF697159 Seymour et al. (1983)
JATV BeAn 423380 Nasua nasua (South American coati) Brazil 1984 Oropouche JQ675601–JQ675603 Figueiredo & Da Rosa (1988)
IQTV IQT9924 Homo sapiens (human) Peru 1999 Oropouche KF697142–KF697144 Aguilar et al. (2011)
Madre de DiosdFMD1303 Homo sapiens (human) Peru 2007 Oropouche KF697145–KF697147 NA
MANV, Manzanilla virus; INGV, Ingwavuma virus; MERV, Mermet virus; BUTV, Buttonwillow virus; FPV, Facey’s Paddock virus; UTIV, Utinga virus; UVV, Utive virus; JATV, Jatobal virus;
IQTV, Iquitos virus.
*Species designations are based on the genetic data presented in this paper.
DPreviously described as an isolate of OYAV. ‘Cat Que virus’ is an unofficial name proposed here to refer to isolate VN 04-2108.
d‘Madre de Dios virus’ is an unofficial name proposed here to refer to isolate FMD1303.
NA, Not applicable.
Manzanilla/Oropouche species complex genomes
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0.04
EU004193 Northway 0234L
HE795099 Sango An5077
JN572074 Taiassui BeAr671
JN801038 Wyeomyia Darien
KF697160 Buttonwillow BFS5002
JN801039 Guaroa BeH22063
EF687077 Jamestown Canyon 61V2235L
EU621833 Chantanga 12718L
HE795096 Sabo IbAn9398
KF697157 Utive PanAn48878
JQ675598 Cat Que VN04-2108
AB297887 Tinaroo CSIRO153
NC001925 Bunyamwera
EU004192 Main Drain BFS5015L
NC005776 Oropouche BeAn19991
EU026160 Maguari BeAr7272L
HM627178 Leanyer NT 16701
HE795087 Aino 38K
JN572071 Sororoca BeAr32149
EF687079 Melao TRVL9375L
KF697147 Madre de Dios FMD1303
HE795093 Peaton CSIRO110
NC004108 La Crosse human/78
AB257769 Ilesha KO-2
JQ675603 Jatobal BeAn423380
EU203678 Snowshoe hare original
AY593726 Mboke DakArY357/6e
AY593728 Ngari DakArD28542/4e
EU789573 Inkoo KN3641L
EU004194 Potosi 89-3380
AF393328 California encephalitis LEIV-11483L
HE649912 Schmallenberg BH80/11-4
JN572062 Anhembi SPAr2984
JN157805 Zungarococha IQE7620
JN572080 Wyeomyia original
JN572068 Macaua BeAr306329
KF697139 Ingwavuma SAAn4165
KF697138 Facey’s Paddock AusCh16129
KF697153 Mermet AV782
KF697150 Manzanilla TRVL3586
JN572077 Tucunduba BeAr278
JN801035 Wyeomyia TRVL8349
AF393326 California encephalitis LEIV-TAH
KF697142 Iquitos IQT9924
HE795090 Douglas 93-6
NC009894 Akabane OBE-1
HE795108 Simbu SAAr53
JN968590 Cachoeira Porteira BeAr328208
NC 022039 Brazoran original
KF697154 Utinga BeAn84785
HE795102 Sathuperi
AB297885 Akabane MP496L
AB257766 Batai MM2222
HE795105 Shamonda IbAn5550
JN572065 Iaco BeAn314206
100
75.4
22.1
100
45.5
90.1
57.2
100
100
100
61.4
100
69.1
100
100
96.2
100
79.5
65.5
100
100
100
100
100
100
76.8
100
90.9
87.2
100
100
100
100
56.4
70.5
100
30.5
100
50.9
100
100
100
58.4
100
100
100
96.1
100
100
94.8
100
61.2
100
Clade A
Clade B
Oropouche
Utinga
Manzanilla
Simbu
Wyeomyia
California
encephalitis
Bunyamwera
Buttonwillow
Facey’s Paddock
Fig. 1. Phylogenetic tree of members of the genus Orthobunyavirus based on the protein-coding portion of the L segment. The
tree was built using translated amino acid sequences in MEGA v5.1 (Tamura et al., 2011) using the neighbour-joining algorithm
and a p-distance matrix. The tree is unrooted and the node labels represent percentage bootstrap support values after 1000
resampling events. Filled circles indicate the genomes that were sequenced in this study. Species designations (left brackets)
are based on the genetic data presented in this paper. Clade labels on the far right correspond to serogroups.
J. T. Ladner and others
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and B ranged from 56.6 to 61.5 %, 32.4 to 39% and 59.7 to
71.9 % for the L, M and S segments, respectively, whilst
similarities within the clades ranged from 65.9 to 99.7 %,
34.4 to 89.1 % and 69.8 to 100 %, respectively (Fig. 2). In
our previous evaluation of LEAV (Savji et al., 2011),
minimum percentage amino acid similarities were pro-
posed as criteria for inclusion of viruses within the same
group, where the term ‘group’ refers to the taxonomic
division currently occupied by serogroups. Clear distinc-
tions were found between intra- and intergroup genetic
similarities at the L and S segments, and cut-offs of 59 and
60 %, respectively, were proposed (Savji et al., 2011). With
currently available data, we saw similarly clear distinctions
in comparisons of L-segment sequences, even when Simbu
serogroup clades A and B were treated separately. In fact,
~77 % (92/120) of the pairwise comparisons between these
two clades fell at or below the proposed 59 % similarity
cut-off, and 90.8 % (109/120) of the pairwise comparisons
were below a similarity cut-off of 60 %, whilst all intraclade
comparisons were well above these cut-offs. In general, we
found levels of intra- and interserogroup genetic similarity
to be less distinct when comparing the S segments, and no
pairwise comparisons between clades A and B met the
previously proposed cut-off for different serogroups.
However, 76.9 % (120/156) of pairwise comparisons
between clades A and B exhibited ,69 % similarity, whilst
all comparisons within the two clades were above this
threshold. Furthermore, this degree of S segment similarity
was on a par with pairwise comparisons between members
of the Wyeomyia and Bunyamwera serogroups (Fig. 2); the
Amino acid similarity (%)Amino acid similarity (%)
Clade B California enceph. Wyeomyia
Clade A
to
clade B
Clade A Clade B California
ence
p
h.
Wyeomyia Bunyamwera Group C
Intra Inter Intra Inter Intra Inter Intra Inter
50 60 70 80 90 100
Clade A
to
clade B
Intra Inter Intra Inter Intra Inter Intra Inter Intra Inter Intra Inter
20 40 60 80 100
Clade A
(a)
(b)
L segment
S segment
Fig. 2. Pairwise genetic similarities (1”amino acid p-distance) among viruses within and between serogroups of
orthobunyaviruses based on the L segment (a) and S segment (b). For the two Simbu serogroup clades, an extra category
is presented that includes only the pairwise similarities between these two groups; this is a subset of the intergroup distances
for both clade A and clade B. See Tables S2 and S3 for the list of sequences used in these analyses.
Manzanilla/Oropouche species complex genomes
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former was not included in our previous analysis (Savji et al.,
2011). Therefore, we argue that the level of evolutionary
divergence between these two Simbu serogroup clades is
more consistent with levels of divergence seen among the
other orthobunyavirus serogroups than that seen within
serogroups.
One of the hallmarks of the Simbu serogroup is its
extensive geographical distribution, and this has been
suggested to be one of the major factors behind the high
level of genetic diversity found within this group (Saeed
et al., 2001a). Even when considered independently, the
two Simbu serogroup clades, both still exhibited extensive
geographical distributions (Fig. S3), which may explain the
high levels of genetic diversity within each of these two
groups compared with many other orthobunyavirus
serogroups (Fig. 2). However, clade A (Manzanilla and
Oropouche species complex viruses) was unique in being
found within the Americas. To date, 77% (10/13) of
Manzanilla and Oropouche species viruses have been
isolated from North and/or South America (including
Inini virus; Saeed et al., 2001a), but no clade B viruses have
been found yet in this region. However, clade A viruses are
not restricted only to the Americas, as isolates of some
representatives have been obtained from Australia, South
Africa and Vietnam (Bryant et al., 2005; Doherty et al., 1979;
McIntosh et al., 1965), and serological evidence exists for the
presence of Ingwavuma virus (INGV) in Nigeria, the Central
African Republic, India, Thailand and Cyprus (http://wwwn.
cdc.gov/arbocat/). These differences in distribution between
the two genetic clades may relate to differences in vector and/
or host range, which can facilitate or restrict the geographical
spread of viruses. More effort on the surveillance and
identification of arthropod vectors of viruses in this group is
necessary to understand better the forces that have shaped
and maintain these distinct distributions.
Species-level phylogenetic relationships
Within clade A, two species complexes have been proposed
(Nichol et al., 2005); however, the genetic data presented
here suggested the presence of at least five different
lineages, which should probably be considered distinct
species (Figs 1, S1 and S2). Based on a limited number of
available sequences, it has been observed that distinct
Orthobunyavirus species tend to differ by at least 10 %
when comparing NP amino acid sequences (S segment).
Utilizing this criterion, clade A should be divided into five
different species with Buttonwillow virus (BUTV) forming
its own species apart from the other Manzanilla species
complex viruses and with the Oropouche species complex
being split into three distinct species. Together, UTIV and
UVV formed one of the new species within the current
Oropouche species complex; the name Utinga is suggested
for this species, as this was the first of its members to be
described (Shope et al., 1967). Facey’s Paddock virus (FPV),
which was a phylogenetic outlier and highly divergent
from all of the other clade A viruses (minimum NP
divergence526 %), represents the other new species pre-
viously attributed to the Oropouche species complex.
These species divisions were phylogenetically consistent in
both the S- and L-segment trees with between-species
amino acid divergences of at least 14.1 and 20.9 %,
respectively, and maximum within-species divergences of
7.3 and 17.3 %, respectively. The M-segment phylogeny
was also generally consistent with these species, with the
exception of the Oropouche species, which was compli-
cated by several potential reassortment events (see below).
Excluding Oropouche species viruses, all M-segment,
amino acid divergences within species were ¡21.4 %,
whilst all divergences between species were ¢32.8 %. These
species were also consistent with serological comparisons
(Tables 2 and 3), and in certain cases corresponded with
available phenotypic information. For example, mosqui-
toes are the primary vectors for most of the Manzanilla
species complex viruses, whereas BUTV has only been
associated with Culicoides midges (http://wwwn.cdc.gov/
arbocat/). Throughout the rest of the paper, we utilize
these five species designations without the use of the term
‘complex’, in order to distinguish them from the species
identified previously by the ICTV (Nichol et al., 2005).
Unassigned viruses
Four of the viruses sequenced here have not been officially
assigned to a species complex by the ICTV. However, using
a combination of genetic (Figs 1, S1 and S2) and serological
data (Tables 2 and 3), it was clear that one of these viruses
(VN04-2108) belonged to the Manzanilla species, whilst
the other three [Iquitos (IQTV), Jatobal (JATV) and
FMD1303]belonged to the Oropouche species. Virus strain
VN 04-2108 was originally reported to be Oya virus
(OYAV), based on indirect immunofluorescence assays and
its high nucleotide similarity to OYAV, based on a partial
S-segment sequence (Bryant et al., 2005). Our S-genome
segment was identical to the sequence from the original
characterization of VN 04-2108; in fact, this sequence is
characteristic of all the ‘Oya’ isolates obtained in Vietnam
during that study (Bryant et al., 2005). However, when
compared now with all of our newly available sequence
data, VN 04-2108 exhibited similar levels of divergence to
four named viruses in the Manzanilla species complex: the
original OYAV isolate (4.1 % amino acid divergence, 9.6 %
nucleotide divergence), Manzanilla virus (MANV, 2.5 %
amino acid divergence, 13.1 % nucleotide divergence),
INGV (5.7 % amino acid divergence, 11.2 % nucleotide
divergence) and Mermet virus (MERV, 4.1 % amino acid
divergence, 14.5 % nucleotide divergence). The prototype
OYAV virus was isolated from a sick pig in Malaysia during
a Nipah virus outbreak and was not available for this study,
so all of these divergences were based only on the published
portion of the S segment available for the original OYAV
isolate (GenBank accession no. AB075611). No genus-wide
framework has been proposed for genetically determining
which orthobunyaviruses should be uniquely named;
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however, based on the levels of genetic divergence among
currently named viruses in this group, VN 04-2108 should
probably be given its own unique name, in which case, Cat
Que virus (CQV) is suggested, as this is the name of the
community in Vietnam where the infected mosquitoes
were collected (Bryant et al., 2005). Alternatively, given the
overall genetic and phylogenetic similarity of VN 04-2108,
MANV, MERV and INGV across all three genome
segments (Figs 1, S1 and S2), it may be more prudent to
simplify the nomenclature by referring to all four simply as
distinct strains of a single named virus, utilizing the name
of the species to which they all belong (e.g. Manzanilla
virus VN 04-2108, Manzanilla virus AV 782). UTIV and
UVV are also strong candidates for synonymization under
a single virus name, as they exhibit even higher levels of
sequence similarity than those within the Manzanilla
species. High levels of genetic similarity are also seen
among named members of the Oropouche species at
particular segments; however, relationships are compli-
cated by patterns of reassortment (see below).
FMD1303 is a previously uncharacterized orthobunyavirus
that was isolated from a febrile human in the Madre de Dios
region of Peru. Serologically, FMD1303 is broadly reactive
with OROV, IQTV and JATV, which suggests that it is a
member of the Oropouche species, and this is consistent
with the available genetic data. The S segment of FMD1303
is identical at the amino acid level to both OROV and IQTV.
This is consistent with what is known about the epidemi-
ology of FMD1303, as OROV and IQTV are the only other
two Simbu serogroup viruses that have been shown to cause
disease in humans (Aguilar et al., 2011; Anderson et al.,
1961). However, based on the nucleotide sequence of the
S segment, FMD1303 falls outside the range of diversity
currently described for OROV/IQTV (Fig. S4). The L-
segment phylogeny is also consistent with this finding,
Table 2. Complement fixation (CF) results
Values are displayed as levels of dilution for antibody/antigen (W, undiluted). Within the family Bunyaviridae, a CF test generally detects NP
antibodies, a marker for the S-segment RNA. In this study, some of the homologous CF titres of four dose hyperimmune ascitic fluid were high
(512–1024), probably explaining the more extensive, low-titre heterologous relationship obtained. MDDV, Madre de Dios virus (an unofficial name
proposed here to refer to isolate FMD1303); CQV, Cat Que virus (an unofficial name proposed here to refer to isolate VN 04-2108); OROV,
Oropouche virus; see Table 1 for other abbreviations. Bold text indicates results from CF tests with antigen and antibody generated from the
same virus.
Antigen Antibody
OROV IQTV MDDV JATV UTIV UVV BUTV CQV INGV MERV MANV FPV
OROV 512/¢32 512/¢8512/¢8128/¢88/¢32 32/¢32 0 32/¢80 16/¢8016/¢8
IQTV 512/¢W512/¢’1024/¢W256/¢’064/¢’064/¢’8/¢’32/¢’016/¢’
MDDV 512/¢’512/¢’1024/¢W256/¢’16/¢’64/¢’064/¢’8/¢’0032/¢’
JATV 512/¢’512/¢’1024/¢8256/¢’032/¢’064/¢’016/¢’08/¢8
UTIV 32/¢32 64/¢832/¢816/¢832/¢32 256/¢32 0 32/¢80 8/¢8032/¢8
UVV 32/¢32 64/¢864/¢816/¢832/¢32 256/¢32 032/¢80 8/¢8016/¢8
BUTV 64/¢864/¢864/¢832/¢80 8/¢832/¢832/¢88/¢88/¢80 8/¢8
CQV 64/¢’64/¢’64/¢832/¢’08/¢’0512/¢’64/¢’512/¢’16/¢’0
INGV 32/¢864/¢864/¢832/¢80 16/¢80512/¢864/¢8512/¢816/¢80
MERV 8/¢832/¢864/¢816/¢80 0 0256/¢832/¢8512/¢88/¢80
MANV 64/¢’64/¢832/¢832/¢80 0 0512/¢’64/¢’512/¢’128/¢’0
FPV 64/¢’32/¢’64/¢W08/¢’32/¢’032/¢’8/¢’00512/¢2
Normal 0 0 0 0 0 0 0 0 0 0 0 0
Table 3. Haemagglutination inhibition results
Haemagglutinating antigen preparation was unsuccessful for BUTV,
MANV, UVV, JATV, IQTV, FPV and MDDV. See Tables 1 and 2 for
abbreviations. Bold text indicates results from tests with antigen and
antibody generated from the same virus.
Antibody Antigen*
OROV UTIV CQV INGV MERV
OROV 1 : 10 240 1 : 160 1 : 80 1 : 20 1 : 40
IQTV 1 : 320 1 : 80 1 : 80 1 : 40 1 : 80
MDDV 1 : 320 1 :80 1 : 80 1 : 40 1 : 40
JATV 0 0 1 : 40 0 0
UTIV 1 : 20 1:20 000
UVV 1 : 80 1 : 80 1 : 40 0 0
BUTV 0 0 1 : 20 0 0
CQV 1 : 160 1 : 80 1 : 2560 1 : 640 1 : 320
INGV 1 : 80 1 : 40 1 : 80 1 : 160 1 : 160
MANV 0 0 1 : 40 1 : 40 1 : 40
MERV 1 : 40 1 : 40 1 : 160 1 : 160 1 : 640
FPV 1 : 20 0 1 : 20 0 0
*1unit is the highest antigen dilution in which complete or almost
complete hemagglutination (HA) occurred. Tests were run with
4units/0.025ml.
Manzanilla/Oropouche species complex genomes
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although for this segment many fewer sequences are
available for comparison, whilst the patterns on the M
segment are complicated by reassortment (see below).
Nonetheless, this virus expands the known diversity of
Oropouche species viruses that infect humans. Due to its
distinctiveness, we suggest that this virus be given its own
name; we propose the name Madre de Dios virus (MDDV),
based on the collection locality.
Partial genome sequences from JATV have previously
demonstrated an association with the Oropouche species
(Saeed et al., 2001a, b), and our genetic and serological
analyses confirm this assignment. However, the complete
genome sequence of JATV (GenBank accession nos
JQ675601–JQ675603) differs from the partial sequences
previously reported for the same strain (GenBank accession
nos AF312380 and AF312382) (Saeed et al., 2001a, b).
These results were confirmed at the Center for Techno-
logical Innovation, Genomic Core, Evandro Chagas
Institute, by obtaining a second full-length sequence of a
lower passage of JATV strain BeAn 423380 (original seed).
Sequences of the original seed and the passaged virus from
the World Reference Center for Emerging Viruses and
Arboviruses collection are identical. The largest discrep-
ancy between the JATV sequences described here and those
that were reported previously lies in the S segment where
the two sequences are only 83.4% identical at the nucleotide
level (90.4 % amino acid identity), with polymorphisms
present throughout the ~700 bases. The M-segment
sequences, on the other hand, are essentially identical across
the 570 bases covered by the partial sequence, except for
several differences at the ends of the partial M sequence. We
cannot account for these differences in the S-segment
sequences, as we could not detect the previously published
sequence in either of our JATV samples, but we are confi-
dent in the quality of the genome sequence reported here.
Reassortment
Reassortment in multi-segmented viruses is a form of
genetic exchange that has the potential to provide many of
the benefits of sexual exchange; for example, the rapid
introduction of novel variation within a lineage, the
uncoupling of beneficial mutations from detrimental
changes and the ability to combine multiple beneficial
mutations that originated in different lineages (Simon-
Loriere & Holmes, 2011). In fact, reassortment has been
implicated in multiple instances of host/vector range shifts
and changes in pathogenicity (Briese et al., 2006; Idris et al.,
2008; Le Noue
¨net al., 2006; Nelson & Holmes, 2007; Parrish
& Kawaoka, 2005; Schrauwen et al., 2011). Laboratory
experiments have demonstrated that reassortment is com-
mon between many bunyaviruses in vitro (Gentsch &
Bishop, 1976; Gentsch et al., 1977, 1980; Iroegbu &
Pringle, 1981; Pringle & Iroegbu, 1982; Reese et al., 2008),
and a number of recent genomic analyses have suggested
that reassortment also plays an important role in viral
evolution in natural populations (Aguilar et al., 2011; Briese
et al., 2006, 2013; Kobayashi et al., 2007; Nunes et al., 2005;
Reese et al., 2008; Yanase et al., 2006, 2010). These examples
include multiple viruses within the genus Orthobunyavirus.
Given the potential evolutionary implications, it is import-
ant to monitor for the prevalence of viral reassortment
events, especially in virus groups known to infect mammals.
To look for reassortment events, nucleotide-level phylo-
genetic analyses were conducted, which included only fully
sequenced members of the five clade A species (Fig. 3).
Phylogenetic discordance, representing potential reassort-
ment, was evident among the phylogenetic trees built from
the three different genome segments. More specifically,
whilst the S and L trees exhibited nearly identical branching
patterns, the M segment supported different relationships
among several of the sequenced viruses. In total, there were
three discrepancies between the M-segment tree and the S/
L-segment trees. The two best-supported discrepancies
(bootstraps ¢75.9 in all trees) involved viruses in the
Oropouche species. The first involved OROV, IQTV and
MDDV. OROV and IQTV were sister taxa in the S/L trees,
whilst IQTV and MDDV were sister taxa in the M tree (Fig.
3). Support for the relationships of these three taxa was
extremely high in all trees (¢99 bootstrap). Sliding-
window analyses in RDP confirmed that these disparate
patterns of divergence were consistent throughout each
genomic segment, as expected with a reassortment event
along one of these three lineages (Figs S5 and S6).
IQTV was recently described as a reassortant between
OROV and an unknown Simbu serogroup virus based on
partial sequences from the S and M segments (Aguilar et al.,
2011). Our results are consistent with this finding;
furthermore, we were able to identify MDDV as a potential
source for the M segment of IQTV. Natural reassortment
between MDDV and OROV is certainly plausible given
their documented geographical distributions: both viruses
have been isolated from the Madre de Dios region of Peru,
and the S segment of IQTV is most similar to the S
segments from clade II of OROV, which is the only clade,
so far, that has been found in Peru (Aguilar et al., 2011;
Saeed et al., 2000). Interestingly, the level of amino acid
divergence between OROV and both IQTV and MDDV
(41–42 %) was on par with levels of divergence seen
between species. Whether this reassortment event has
resulted in any changes in virulence, vectors or range has
yet to be determined. It is also important to keep in mind
that, with the current available data, it is impossible to
know for certain which of these three viruses represents the
true reassortant (Briese et al., 2013). The addition of more
complete genome sequences from each of these viral
lineages should clarify relationships.
The second potential case of reassortment involved JATV,
the Utinga species (i.e. UTIV/UVV) and the lineage leading
to the three human viruses (OROV, IQTV and MDDV). In
the L and S trees, JATV formed a clade with the three
human viruses, whereas in the M-segment tree JATV
formed a well-supported clade with the Utinga species (Fig.
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3). The RDP analyses demonstrated that these discordant
relationships were consistent throughout the entirety of
each genome segment (Figs S5 and S6). Based on the high
levels of sequence divergence among all three groups of
viruses (.45 %), this reassortment event is likely to have
occurred many generations ago or to have involved parental
viruses that have yet to be isolated and/or sequenced.
JATV has been reported previously to be a reassortant with
an S segment from OROV and an M segment from an
uncharacterized virus (Saeed et al., 2001b). Whilst our
findings were generally consistent with the overall conclu-
sion of reassortment, our S-segment sequence was not
consistent with the previously reported S segment (Saeed
et al., 2001b). The S segment in this previous publication
fell within the OROV clade, whereas our S sequence was a
phylogenetic outgroup to the S segments of OROV, IQTV
and MDDV (Fig. 3). Our L segment is also consistent with
this placement, demonstrating that this is an S/L- vs M-
segment reassortment event. Also, based on the available
data, it was again impossible to distinguish which of the
viral lineages involved represents the true reassortant.
The third potential reassortment event involves Manzanilla
species viruses. CQV and INGV were sister taxa in the L/S
trees, whereas CQV formed a clade with MANV and
MERV in the M-segment tree (Fig. 3). The bootstrap
support for these different relationships, however, was
lower than that seen in the other discrepancies and the RDP
analyses demonstrated that the divergence signals were not
consistent across the genome segments (Figs S5 and S6).
Therefore, it is unclear whether this third discrepancy is
due to reassortment or simply the result of ambiguity in
the patterns of divergence.
Conclusion
The addition of 11 fully sequenced genomes for viruses in
the Manzanilla and Oropouche species complexes has
highlighted a deep evolutionary divide between these two
species complexes and the rest of the Simbu serogroup.
With sequence data from all three genome segments, we
found compelling evidence to divide these two species
complexes into five distinct species, and we were also able
(a) L segment
0.04
JQ675603 Jatobal BeAn423380
KF697160 Buttonwillow BFS5002
KF697150 Manzanilla TRVL3587
KF697139 Ingwavuma SAAn4165
KF697147 Madre de Dios FMD1303
JQ675598 Cat Que VN04-2108
KF697153 Mermet AV782
KF697142 Iquitos IQT9924
KF697138 Faceys Paddock AusCh16129
NC005776 Oropouche BeAn19991
KF697157 Utive PanAn48878
KF697154 Utinga BeAn84785
100
100
100
90.5
100
100
80.5
100
100
100
(b) S segment
0.04
KF697144 Iquitos IQT9924
JQ675600 Cat Que VN04-2108
KF697156 Utinga BeAn84785
KF697148 Manzanilla TRVL3587
KF697162 Buttonwillow BFS5002
KF697146 Madre de Dios FMD1303
NC005777 Oropouche BeAn19991
JQ675601 Jatobal BeAn423380
KF697158 Utive PanAn48878
KF697152 Mermet AV782
KF697136 Faceys Paddock AusCh16129
KF697141 Ingwavuma SAAn4165
75.4
100
39.5
99.7
99.5
100
100
99.5
38.6
99.5
(c) M segment
0.05
KF697137 Faceys Padock AusCh16129
JQ675602 Jatobal BeAn423380
KF697159 Utive PanAn48878
KF697140 Ingwavuma SAAn4165
JQ675599 Cat Que VN04-2108
KF697161 Buttonwillow BFS5002
KF697149 Manzanilla TRVL3587
KF697138 Mermet AV782
NC005775 Oropouche BeAn19991
KF697155 Utinga BeAn84785
KF697145 Madre de Dios FMD1303
KF697143 Iquitos IQT9924
100
98.9
100
100
75.9
100
100
97.2
56.5
98.9
Fig. 3. Nucleotide-level phylogenetic trees including only the fully sequenced members of the Oropouche and Manzanilla
species complexes. All trees were built in MEGA v5.1 (Tamura et al., 2011) using the maximum-likelihood framework with partial
deletions. The trees are unrooted. Node labels represent percentage bootstrap support values after 1000 resampling events.
Manzanilla/Oropouche species complex genomes
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to identify three potential reassortment events among
viruses in these species. Two of these involved viruses that
infect humans, and levels of sequence divergence on the
reassorted segment were on a par with divergences seen
between species. Future work is needed to determine
whether any of these reassortments have affected virulence.
METHODS
Virus isolates. All virus stocks used in this study were obtained from
the World Reference Center for Emerging Viruses and Arboviruses at
the University of Texas Medical Branch (UTMB). The JATV original
seed was provided by the World Health Organization Reference
Centre for Arboviruses at the Department of Arbovirology and
Hemorrhagic Fevers, Instituto Evandro Chagas, Brazilian Ministry of
Health. Virus strain FMD1303 was originally isolated at the US Naval
Medical Research Unit No. 6 (NAMRU-6) in Lima from a blood
sample obtained from a febrile human in Madre de Dios Department,
Peru, on 22 March 2007. The histories of the other isolates sequenced
in this study have been published previously (see Table 1).
Serological characterization. All the sequenced viruses were
compared with each other and with OROV for serological similarity.
Methods used to prepare antigens for the complement fixation (CF)
tests and for the preparation of immune ascitic fluids have been
described previously (Beaty et al., 1989; Travassos da Rosa et al., 1983;
Xu et al., 2007). Both antigens and antibodies were produced in mice.
CF tests were performed by the microtitre technique (Beaty et al.,
1989; Xu et al., 2007), using 2 U of guinea pig complement with
overnight incubation of the antigen and antibody at 4 uC. CF titres
were recorded as the highest dilutions giving 3+or 4+fixation of
complement. Titres of 1 : 8 or greater were considered positive.
Haemagglutination inhibition (HI) testing was performed in micro-
titre plates, as described previously (Travassos da Rosa et al., 1983).
HI tests were performed with four haemagglutination units of virus at
the optimal pH (5.75) against serial twofold antiserum dilutions,
starting at 1 : 20. HI titres of 1 : 20 or greater were considered positive.
CF and HI tests were performed at the UTMB, Galveston, TX, USA.
Genome sequencing. The BeAn 423380 (JATV) and VN 04-2108
(CQV) strains were sequenced and assembled at the Center for
Infection and Immunity, Columbia University. The JATV original
seed was sequenced and assembled at the Center for Technological
Innovation, Genomic and Bioinformatic Cores, Evandro Chagas
Institute, Brazil. For these strains, total RNA was first extracted from
viral supernatant preserved in TRIzol LS (Invitrogen) and then
treated with DNase I (DNA-Free; Ambion). cDNA was generated
using the Superscript II system (Invitrogen) with random hexamers
linked to an arbitrary 17-mer primer sequence (Cox-Foster et al.,
2007). The resulting cDNA was treated with RNase H and then
amplified by random PCR (Cox-Foster et al., 2007). Products longer
than 70 bp were selected by column purification (MinElute: Qiagen)
and ligated to specific adapters for sequencing on the 454 Genome
Sequencer FLX (454 Life Sciences) without fragmentation of the
cDNA (Cox-Foster et al., 2007; Margulies et al., 2005; Palacios et al.,
2008). Software programs accessible through the analysis applications
at the GreenePortal website were used for removal of primer
sequences, redundancy filtering and sequence assembly. These
genomes were completely confirmed using dye-labelled, dideoxynu-
cleotide sequencing.
All other strains were processed at the Center for Genome Sciences, US
Army Medical Research Institute of Infectious Diseases (USAMRIID),
Fort Detrick, MS, USA. For these strains, total RNA was extracted from
viral supernatant preserved in TRIzol LS and was amplified using
sequence-independent single primer amplification, as described
previously (Djikeng et al., 2008). Amplicons were sheared to ~400 bp
and used as starting material for Illumina TRUseq DNA libraries.
Sequencing was performed on a HiSeq 2500. Primers were trimmed
from the sequencing reads using Cutadapt (Martin, 2011), quality
filtering was conducted with Prinseq-lite (Schmieder & Edwards, 2011)
and then genomes were assembled using Ray Meta (Boisvert et al.,
2012) in combination with custom scripts. When necessary, terminal
sequences were completed through PCR and dideoxynucleotide
sequencing using a universal orthobunyavirus primer targeting the
conserved viral termini (59-AGTAGTGTRC-39) in combination with
specific primers designed from the sequences generated from the de
novo assembly. In addition, four genomes were confirmed with
dideoxynucleotide sequencing (BUTV, FPV, UTIV and UVV). These
included the six genome segments with the lowest levels of sequence
coverage (630 to 6767). These sequences confirmed the high-quality
of assemblies achieved through these methods.
Phylogenetic analysis. Separate phylogenetic analyses were con-
ducted for each of the three genome segments using only the protein
coding portions of the genome. Orthobunyavirus sequences from
GenBank were included to provide a representative picture of the
entire genus; many of the sequences included cover only a portion of
the coding region. Sequences were aligned using the CLUSTAL
algorithm, which was implemented at the amino acid level in MEGA
v5.1 (Tamura et al., 2011) with additional manual editing to ensure
the highest possible alignment quality. Neighbour-joining analyses
using p-distance at the amino acid level were performed. The
statistical significance of the tree topology was evaluated by 1000
replications of bootstrap resampling. Phylogenetic analyses were
performed using MEGA v5.1 (Tamura et al., 2011).
Reassortment analysis. To identify potential reassortment events,
the data were mined for evidence of phylogenetic discordance. For
this analysis, additional phylogenetic trees were reconstructed, which
included only fully sequenced members of the Oropouche and
Manzanilla species complexes. These trees were reconstructed from
the same alignments used above; however, to provide additional
power, these trees were conducted using a maximum-likelihood
framework at the nucleotide level [implemented in MEGA v5.1
(Tamura et al., 2011) with the Tamura–Nei substitution model,
partial deletion, uniform rates among sites and 1000 bootstrap
replications]. Potential reassortment events were then verified using
the manual BOOTSCAN (Martin et al., 2005) and distance plot methods
in RDP4 (Martin et al., 2010).
Pairwise sequence analysis. Pairwise sequence divergences were
calculated among each of our 11 viruses and all of the other
orthobunyaviruses with complete genome segment sequences using
MEGA v5.1 with pairwise deletions (Tamura et al., 2011). For the
comparisons of divergence within and between serogroups, only one
representative of each named species was utilized. This downsampling
was done to avoid bias due to intensive sampling of certain viruses.
ACKNOWLEDGEMENTS
We would like to thank Carolina Guevara and staff of the NAMRU-6
Virology Department, Lima, Peru, for providing MDDV (FMD1303)
and Clayton Lima, Keley Nunes and Jedson Cardoso, Genomic and
Bioinformatic Cores, Center for Technological Innovation, Evandro
Chagas Institute, for sequencing and assembling the JATV original
seed. The work at USAMRIID was funded by the Defense Threat
Reduction Agency project no. 1881290. Work at UTMB was supported
by National Institutes of Health contract HHSN27220100004OI/
HHSN27200004/D04. Sequencing and assembling of the JATV original
seed was supported by Evandro Chagas Institute internal grant, and
J. T. Ladner and others
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CNPq grants 3016401/2010-2 and 302032/2011-8. Work in the Center
for Infection and Immunity was supported by the National Institutes of
Health (AI057158), USAID PREDICT and the Department of Defense.
We are grateful to the Peruvian Ministry of Health for supporting the
study and the physicians at the study site for their participation and
help. The study protocol 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. Opinions, interpretations, conclusions
and recommendations are those of the authors and are not necessarily
endorsed by the US Army, Department of the Navy, Department of
Defense, nor the U.S. Government.
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