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Arch Virol (2007) 152: 2237–2247DOI 10.1007/s00705-007-1069-zPrinted in The Netherlands
Natural M-segment reassortment in Potosi and Main Drain viruses:implications for the evolution of orthobunyaviruses
T. Briese, V. Kapoor, W. I. Lipkin
Center for Infection and Immunity, Mailman School of Public Health, Columbia University, New York, NY, USA
Received 3 March 2007; Accepted 31 August 2007; Published online 23 September 2007# Springer-Verlag 2007
Summary
Recently, we identified Batai virus as the M-segmentreassortment partner of Ngari virus. Extension ofgenetic analyses to other orthobunyaviruses relatedto the Bunyamwera serogroup indicates additionalnatural genome reassortments. Whereas the relativephylogenetic positions of all three genome segmentsequences were similar for Northway and Kairi vi-ruses, the relative positions of Potosi and MainDrain virus M-segment sequences diverged fromthose of their S- and L-segments. Our findings in-dicate M-segment reassortment in Potosi and MainDrain viruses and demonstrate natural genomereassortment as a driving force in the evolution ofviruses of the Bunyamwera serogroup.
Introduction
The Bunyaviridae are among the largest viral fami-lies, with over 300 members classified in the fivegenera Orthobunyavirus, Nairovirus, Phlebovirus,Hantavirus, and Tospovirus (ICTVdb http:==phene.
cpmc.columbia.edu [51]). They are unified by acommon morphology that features a segmented ri-bonucleoprotein engulfed by a lipid envelope that isdecorated with small glycoprotein spikes, formingmainly spherical particles of 80–120 nm; nonethe-less, the family is heterogeneous with respect tohost range and transmission mode. Whereas tospo-viruses infect plants, members of the other fourgenera are animal viruses [51]. In addition, whilethe family encompasses the majority of describedarthropod-borne viruses (arboviruses) transmittedby vectors such as thrips (e.g., tospoviruses), ticks(e.g., nairoviruses, phleboviruses), biting flies (e.g.,phleboviruses, nairoviruses) and mosquitoes (e.g.,orthobunyaviruses, phleboviruses), the members ofthe genus Hantavirus are not known to infectarthropods and are maintained in rodent reservoirs,adapted to a particular host species [42].Although aspects of genome organization vary
among genera, a distinctive family feature is thepresence of a tripartite single-stranded RNA ge-nome that encodes replicase functions by the largesegment (L-segment), two surface glycoproteins bythe medium size segment (M-segment), and a nucle-ocapsid protein (N) by the small genome segment(S-segment) [51]. Among orthobunyaviruses, theM-segment codes for a cotranslationally processedpolyprotein that comprises the N- and the C-ter-
Correspondence: Thomas Briese, Center for Infectionand Immunity, 1801 Mailman School of Public Health,Columbia University, New York, NY 10032, USAe-mail: [email protected]
minal glycoproteins, GN and GC, separated by asmall nonstructural membrane protein, NSm [23].According to recent findings, NSm participates invirus assembly [52]. Another nonstructural protein,NSs, coded by a second open reading frame (ORF)of the orthobunyaviral S-segment, has been shownto counteract the innate host immune response byblocking alpha=beta interferon induction [7, 53].The segmented genome structure affords these
viruses an opportunity for genome segment reassort-ment during a mixed infection event. Although ge-nome reassortment between viruses of the Californiaencephalitis (CE) or the Bunyamwera (BUN) sero-group occurs readily in experimental settings [4, 6,26, 29, 48], natural reassortants are only infre-quently reported [27, 32]. Genetic analyses recentlyidentified UgMP-6830 from Uganda as an isolateof Batai virus (BATV) and indicated that its M-segment most closely matched that of Ngari virus(NRIV), suggesting a historical M-segment reas-sortment event [10]. Here, we present and analyzeS-, M-, and L-segment sequences of four other ortho-bunyaviruses: Potosi virus (POTV) and Northwayvirus (NORV), two North American viruses vec-tored chiefly through Aedes mosquitoes within theirdeer or rodent reservoir, respectively; Main Drainvirus (MDV), vectored by Culicoides midges, butalso mosquitoes of the Aedes and Culiseta genera,between North American rodent reservoir hosts;and Kairi virus (KRIV), vectored predominantlyby Aedes and Wyeomyia mosquitoes in largely un-defined reservoirs throughout South and MiddleAmerica. All four viruses have been subsumed inthe Bunyamwera serogroup, although KRIV andMDV are serologically more distantly related toother members of the group and are considered toform separate serocomplexes [12]. Phylogeneticanalysis of sequences from all three genome seg-ments suggests that POTV and MDV represent nat-ural reassortment events.
Materials and methods
Bioinformatics and primer design
Full-length M-segment sequences for orthobunyaviruses ofthe CE and BUN serogroups and Guaroa virus were retrievedfrom GenBank and analyzed for conservation. Primers in
highly conserved domains were chosen according to com-mon standards including avoidance of stable stem-loops,primer-dimer formation, and strong 30-terminal hybridiza-tion. Sequence regions devoid of stable secondary structurewere sought for primer selection, however, compromises foreither one of these attributes had to be made for individualpermutations of degenerate primers. We recently describedGreene SCPrimer, a software tool that in part integrates de-sign constraints for selecting degenerate primers from mul-tiple sequence alignments (http:==scprimer.cpmc.columbia.edu [30]).
Virus isolates and nucleic acid extraction
Stocks of Potosi virus (POTV) strain 89–3380, Northwayvirus (NORV) strain 0234, Main Drain virus (MDV) strainBFS5015, and Kairi virus (KRIV) strain TRVL8900 werekindly provided by Robert Lanciotti, Robert Tesh, and thelate Robert Shope. Eighty microliters of virus stock wasextracted with Tri-Reagent following the manufacturer’s pro-tocol (MRC, Cincinnati, OH). Total RNA was dissolved in20ml RNase-free H2O.
Reverse transcription – polymerase chain reaction(RT-PCR) and sequencing
Three-microliter aliquots of total RNA were reverse tran-scribed with random hexamers (Amersham PharmaciaBiotech, Uppsala, Sweden) in a 20-ml volume by using theSuperscript II system (Invitrogen, Carlsbad, CA). PCR am-plification [49] with various primer pairs was performed byincubating 0.5ml cDNA, dNTP (200mM), MgCl2 (3.0mM),primers (1.6mM, each), and Bio-X-act polymerase (1.6 units)in 25ml 1! Opti buffer (supplied with polymerase; Bioline,London, UK) for 45 cycles in a PTC-200 thermocycler(MJ Research, Waltham, MA), applying a cycling protocolof 92 "C for 1min, 47 "C, 48 "C, or 52 "C for 1min (seeTable 1), and 68 "C for 1min, followed by a final extensionfor 10min at 68 "C. Amplification products were size-frac-tionated in 1.3% agarose gels and visualized by ethidiumbromide staining at the end of the run. Products were elutedfrom gel fragments and sequenced either directly, result-ing in a majority sequence, or where necessary after clon-ing into pGEM-Teasy plasmid vector (Promega, Madison,WI). Cloned sequences were obtained from at least threeplasmids for both strands by automated dideoxy-sequenc-ing [50] using BigDye Terminator Cycle Sequencing kitson ABI Prism Genetic Analyzer systems (Applied Bio-sytems, Foster City, CA).
Sequence data generated by this work are available atGenBank under accession numbers AY729652, andEU004186-EU004194.
Sequence analyses and phylogenetic analysis
Programs of the Wisconsin GCG Package (Accelrys, SanDiego, CA) were used for sequence assembly and analysis;
2238 T. Briese et al.
percent sequence identities were calculated using ‘gap’ atdefault settings. Topology and targeting predictions wereobtained by employing SignalP-NN=SignalP-HMM, NetN-Glyc, TMHMM (http:==www.cbs.dtu.dk=services), the web-based version of TopPred2 (http:==bioweb.pasteur.fr=seqanal=interfaces=toppred.html), and Phobius (http:==phobius.cgb.ki.se=index.html) [20, 31, 34, 43, 44]. Phyloge-netic analyses were performed by using MEGA 3.1 software[35].
Results
Acquisition of POTV sequence
Although POTV is reported to be indistinguishablefrom Cache Valley virus (CVV) in a cell-lysate an-tigen enzyme-linked immunosorbent assay (ELISA)[3], it is readily differentiated from CVV in cross-neutralization tests. The latter finding is in line withthe divergence of a POTV GN sequence fragmentfrom that of CVV [3, 27]. In an attempt to identifythe genetic basis for the observed reaction pattern,we determined and analyzed sequences from allthree POTV genome segments.POTV sequence was amplified by RT-PCR using
consensus primers that target highly conserved do-mains in the genomes of viruses of the CE and
BUN serogroups (Table 1 and Ref. [8]). Nucleotidesequence was determined directly from amplifica-tion products if permitted by size and quantity, orafter cloning into a plasmid vector. To test the va-lidity of initial draft sequences and to generate se-quence for intervening regions not covered byproducts obtained with consensus primers, we useddraft sequence to design POTV sequence-specificprimers (primer sequences available upon request).The assembled M-segment consensus indicatesa 1438-amino-acid (aa) coding sequence for thePOTV polyprotein that shows over its entire lengthconsiderable divergence from the CVV M-segmentsequence (60% nucleotide (nt) sequence and 56%aa sequence identity; Fig. 1).Interestingly, the use of amplification primers
outside of nt positions 1079–2169 of the POTVM-segment sequence yielded shorter products thanexpected; products of only the expected size wereobtained when one primer was contained withinthis region. Similar results were not observed withNORV, MDVor KRIV (see ‘Comparative sequenceanalyses’, below). Analysis of the amplificationproducts was compatible with the presence of adeletion between nt 1079 and 2169, correspondingto aa positions 340–704 (Fig. 1). This observation is
Table 1. PCR amplification primers
Primer Sequencea 30-position Annealingtemperature
SBUNS-5-U-6 50-CGGCGCC AGT AGT GTA CTC CAC 947 48 "CBUNS-3-L947 50-GCGGCC AGT AGT GTG CTC CAC 15Cac-2-FWD 50-dCT TAA CyT TGG rGG CTG GA 290 52 "CCac-7-REV 50-CTv ACr TTd Gyy TTC TTC CA 722
MBUN-S5-F 50-GCCGC AGT AGT GTA CTA CCG ATA yA 20 48 "CM940C-R 50-CTr GCw GCT CTw AGr CTT TTr TAm CC 936BUN-M-IXf 50-TGG GGn yGy GAr GAr Twy GG 3247 47 "CBUN-M-XIIr 50-TTk GTy TTT TGk ACA TTk CC 3543M3560-F 50-TCn AAr GGh TGy GGn AAT GT 3550 47 "CBUN-S3-R 50-CGCGCC AGT AGT GTG CTA CC 4445
LBUNL-5n 50-CGCCGC AGT AGT GTA CTy CTA 15 48 "CBUNL650R 50-ACC AkG GTG CTG TmA rAG TGA ArT CwC CAT 601
a Non-authentic bases added to some primers are indicated by italics.Nucleotide positions refer to type species Bunyamwera virus, prototype 1943, Acc. No. D00353, M11852, and X14383.
Genetic analyses identify Potosi and Main Drain viruses as reassortant viruses 2239
Fig.1.
Multiple
sequence
alignmentofM-segmentsequencesfrom
virusesrelatedto
theBUN
serogroupoforthobunyaviruses.NXT=S
potential
N-glycosyla-
tionsite(greyshading);___potentialtransm
embraneregionsforpredictedsignalpeptide(S)ormem
braneanchor(A);xxxconserved
proteolyticcleavagemotif
atC-terminusofGN;tttconserved
potentialtrypsincleavagesite[24];
----
-proposedfusionpeptidedomain[46];#conserved
cysteineresidue;
deletion
identified
forPOTV
betweennt1079(N
Sm)and2169(G
C);Consconsensussequence
2240 T. Briese et al.
Tab
le2.
S-Segmentsequence
conservationbetweenPOTVandother
orthobunyaviruses
%Aminoacid
identity
b%
Nucleotideidentity
b
N=(NSs)
CVV
POTV
NORV
MAGV
MDV
BATV
AUSa
BUNV
NRIV
ILEV
MBOV
GERV
KRIV
GROV
CVV
88.8
86.5
84.6
82.4
82.7
84.3
80.6
79.0
77.7
78.5
71.2
66.7
62.1
POTV
98.7
(100)
86.8
83.6
83.3
82.0
83.0
81.1
80.6
79.1
79.6
71.3
67.4
63.9
NORV
96.2
(96.1)
94.9
(96.1)
86.3
84.2
80.8
84.4
80.2
79.2
79.8
80.2
73.2
67.0
63.4
MAGV
95.7
(97.1)
95.3
(97.1)
94.9
(95.1)
82.4
81.3
81.8
81.4
79.1
79.7
79.3
69.8
66.0
65.0
MDV
89.7
(94.1)
89.3
(94.1)
91.9
(95.1)
91.5
(93.1)
78.3
80.1
79.6
78.4
76.4
76.0
69.3
68.0
63.6
BATV
94.0
(94.1)
93.6
(94.1)
94.9
(94.1)
93.2
(95.1)
89.3
(90.2)
86.6
78.0
78.9
76.4
76.9
72.3
63.8
62.7
AUSa
95.3
(98.0)
94.9
(98.0)
95.3
(94.1)
92.7
(95.1)
88.9
(92.2)
97.4
(94.1)
80.0
79.1
78.4
78.2
72.0
68.8
61.1
BUNV
90.6
(88.2)
91.0
(88.2)
92.7
(88.2)
91.0
(88.2)
90.2
(86.3)
91.9
(88.2)
92.7
(86.3)
95.1
85.0
85.1
72.9
68.1
61.8
NRIV
89.3
(88.0)
89.7
(88.0)
92.8
(89.1)
91.0
(88.0)
89.2
(87.0)
92.4
(89.1)
92.8
(85.9)
99.6
(100)
85.5
85.5
70.2
67.8
62.4
ILEV
90.2
(87.3)
89.7
(87.3)
90.2
(85.3)
88.5
(85.3)
86.8
(85.3)
89.3
(85.3)
90.6
(85.3)
94.0
(95.1)
93.3
(94.6)
98.0
69.1
66.7
63.0
MBOV
90.2
(88.2)
89.7
(88.2)
90.2
(86.3)
88.5
(86.3)
86.8
(84.3)
89.7
(86.3)
90.6
(86.3)
94.0
(94.1)
93.7
(93.5)
99.2
(99.0)
69.1
66.7
61.4
GERV
75.2
(70.3)
74.4
(70.3)
76.1
(67.3)
73.9
(68.3)
74.4
(66.3)
77.4
(67.3)
76.9
(69.3)
75.2
(70.3)
74.9
(70.3)
74.8
(70.3)
75.2
(70.3)
62.8
58.3
KRIV
69.2
(68.6)
69.2
(68.6)
69.7
(69.6)
68.0
(67.7)
70.5
(68.3)
68.4
(66.7)
69.2
(67.7)
70.9
(66.7)
71.3
(67.4)
71.8
(65.7)
71.8
(65.7)
62.4
(56.0)
64.7
GROV
70.9
(42.9)
70.9
(42.9)
70.1
(44.1)
69.2
(41.7)
68.8
(46.4)
70.5
(42.9)
70.5
(42.9)
68.8
(41.7)
69.5
(44.6)
68.0
(44.1)
68.4
(42.9)
63.7
(41.7)
69.7
(50.0)
aUncharacterizedAustralian
Bunyam
weravirusisolate
AF325122.
bNucleotideconservationisindicated
intheupperrighthalfofthetable.A
minoacidconservationisindicated
inthelowerlefthalfofthetable;N
sequences,plain
text;NSssequences,textin
parentheses.
Genetic analyses identify Potosi and Main Drain viruses as reassortant viruses 2241
reminiscent of a finding in Maguari virus (MAGV)revertants, where similar deletions spanning theNSm and N-terminal GC region were generatedduring reversion from a temperature-sensitive mu-tant phenotype [47]. The presence of NSm-deletedas well as sequences co-linear to other ortho-bunyaviral genome sequences reveals a notablegenomic sequence heterogeneity of POTV stockvirus.The S-segment sequence of POTV was similar
in length to other BUN S-segment sequences [22],encoding an N protein of 233 aa and an NSs proteinof 101 aa that is translated from an ORF overlap-ping in the þ1 frame with that of N. Remarkably,the POTV S-segment sequence shares a high de-gree of sequence identity with that of CVV at thent level (89%) as well as at the aa level for boththe N and the NSs ORFs (99% and 100%, respec-tively) (Table 2).Last, we amplified a portion of the POTV L-
segment. The sequence had less than 74% se-quence identity at the nt level (81% at the aa level)to BATV, the most closely related orthobunyavirusL-segment sequence available in GenBank at thetime of this analysis (data not shown).In an effort to identify sequences closely related
to the M- and L-segment sequences determined forPOTV, we applied our panel of consensus primersto PCR amplification of corresponding sequencesfrom NORV, MDVand KRIV, using the same strat-egy as outlined for POTV.
Comparative sequence analyses
Analysis of POTV, NORV, MDVand KRIV M-seg-ment sequence indicated a highly divergent lengthand sequence of their untranslated regions (UTRs)outside of the conserved segment termini. Closesequence homology was observed only betweenthe UTRs of CVV and MAGV (data not shown),and those of BATV and NRIV [10]. The codingsequences showed the common orthobunyavirusorganization with mature proteins in the order GN-NSm-GC. Topological analyses of POTV, NORV,MDV, and KRIV coding sequence indicate thatNSm has a luminal N-, and a cytoplasmic C-termi-nal domain framed by two potential internal signal
peptide sequences (Fig. 1). This structure is consis-tent with earlier bioinformatics predictions basedon Guaroa virus (GROV) M-segment sequence [8]and experimental data recently obtained for theNSm of Bunyamwera virus (BUNV) [52]. The ex-tended set of available sequences indicates conser-vation of only three potential N-glycosylation sites:an N- and a C-terminal site in GN, and a single sitelocated C-terminally in GC (Fig. 1). Other potentialglycosylation sites appear to follow more type-spe-cific patterns. Sequence conservation for GN ishigher than for NSm or GC and is most pronouncedin a region between the conserved C-terminal gly-cosylation site and the terminal protease cleavagemotif KSLRV=AAR [24]. The topological modelfor NSm predicts that this region has a cytoplasmiclocation. Conservation is also noted for a motifin the cytoplasmic domain of NSm, and for threeC-terminal motifs located around the conservedglycosylation motif of GC (Fig. 1), including a pro-posed fusion peptide domain [46]. Highest sequencevariability is found in an approximately 80-aa regionpreceding the conserved potential trypsin recogni-tion site in GC [24].
Phylogenetic analysis
The reconstructed phylogeny based on S-segmentnt sequence identifies POTV, together with NORV,as the closest matches to the CVV sequence. Theoverall topology was consistent, irrespective of theanalysis model applied; Fig. 2A shows a consen-sus tree of 1000 bootstrap repetitions applying aTamura-Nei Neighbor-Joining model. Integratingrecently determined S-segment sequences did notsignificantly change the tree topology compared tothat reported by Dunn et al. [22]. However, theadditional sequences did indicate distinct geneticclades within the serogroup. A CVV-related cladeincludes CVV, POTV, NORV, MAGV, and MDV, aswell as more distantly related the BATV sequences.A BUNV-related clade encompasses the Africanviruses BUNV=NRIV, Ilesha (ILEV) and Mboke(MBOV), which separate from the phylogeneticallymore distant Germiston virus (GERV), KRIV, andGROV sequences (Fig. 2A). Although relationshipsfor N and NSs aa sequences are similar to those
2242 T. Briese et al.
Fig.2.
Phylogenetic
analysisofselected
orthobunyavirusS-,M-,andL-segmentsequences.Phylogenetic
treesbased
on(A
)S-,(B
)M-,and(C
)partial
L-
segmentnucleotidesequence
(approx.570nt)werereconstructed
bytheNeighbor-JoiningmethodapplyingaTam
ura-N
eimodel
with1000pseudoreplicates;
boostrapvalues
above60%
areshow
nat
therespective
branches.GenBankaccessionnumbersfortherespective
sequencesareindicated.California
serogroup
virusesSerra
doNavio
(SDNV),LaCrosse(LACV),andprototypeCalifornia
encephalitis
(CEV),as
wellas
Sim
buserogroupvirusOropouche(O
ROV),are
included
forcomparison.Shadingindicates
virusesanalyzedin
this
study
Genetic analyses identify Potosi and Main Drain viruses as reassortant viruses 2243
observed with nt sequence, the MDV aa sequencesare placed by some software models in a closer re-lationship to Asian and African sequences (data notshown [22]).The overall topology of phylogenetic trees ob-
tained with M-segment sequences and S-segmentsequences is similar; however, POTV and MDVare more closely related to KRIV than to CVV intheir M-segment sequence (Fig. 2A, B). Interestingly,although MAGV is considered to be a subtype ofCVV [14], POTV and CVVare closer in S-segmentsequence than are CVV and MAGV. Compared tothat, POTV and KRIV appear to be less similar intheir M-segment sequence. Phylogenetic analysisof M-segment sequences indicates a closer relation-ship between GERVand BUNV, and between ILEVand the CVV clade than indicated by the S-segmentsequence analysis.Given the highly divergent relationships ob-
served for the S- and M-segment sequences ofPOTV and MDV, it was of interest to assess howtheir L-segment sequences would match with thatof CVVor KRIV. Thus, we obtained L-segment se-quence of CVV strain 6V633, as well as of MAGVstrain BeAr7272, for a region corresponding toL-segment sequences reported for other BUN ser-ogroup viruses. Although the absence of a largesequence repertoire precludes phylogenetic analy-sis as comprehensive as that achieved for S andM segments, the POTV and MDV L-segment se-quences appear more closely related to CVV,MAGV and NORV than to KIRV (Fig. 2C).
Discussion
Phylogenetic analysis of sequences from all threegenome segments of POTV, NORV, MDV, CVV,MAGVand KRIV indicates a divergent phylogenet-ic relationship for POTV and MDV M-segmentsequences in comparison to that of their S- andL-segment sequences. In contrast, consistent rela-tive phylogenetic relationships are observed for allthree genome segment sequences of NORV andKRIV, as well as CVV and MAGV. These findingssuggest at least one M-segment reassortment eventin the evolutionary history of POTV and MDV, in-cluding possibly a secondary reassortment of one
of the M-segments to generate the second of thesetwo reassortant viruses.POTV was first isolated in 1989 when Aedes
albopictus mosquitoes from Potosi, Missouri,were investigated for arbovirus infection [25]. Ae.albopictus mosquitoes, native to Asia, had beendetected in 1985 in Houston, Texas, and the appear-ance of Ae. albopictus larvae the subsequent yearin imported tire casings at the port of Seattle,Washington, suggested a potential route for intro-duction of this new vector to the United States.However, lack of vertical transmission of POTVby Ae. albopictus mosquitoes argued against an in-troduction of the new virus into the Potosi areathrough infected eggs [38]; furthermore, cross-neu-tralization tests did not indicate a relationship ofPOTV to Asian BATV. Indeed, the original reportof POTV describes an inefficient neutralization byKRIV antibodies [25], a result in line with the se-quence findings reported here.Molecular studies indicate that POTV has fre-
quently been misidentified in ELISA as CVV, oreven Jamestown Canyon virus [3], and the develop-ment of specific detection assays suggest thatPOTV has a much wider distribution than previous-ly anticipated [3, 41]. These findings and our ownemphasize the complementing role of moleculardiagnostics and highlight the importance of im-plementing broad-range diagnostic tools such asmultiplex PCR or microarray platforms that arecapable of considering not only one suspected path-ogen, but interrogate a wide range of potential can-didates simultaneously [9, 36, 45].Since the isolation of CVV in 1956 [28], other
newly discovered orthobunyaviruses considered tobe native to North America include MDV, isolatedfrom midges collected in California in 1964 [11],and NORV, identified in mosquitoes and sentinelrabbits at Northway, Alaska, in 1970=71 [18]. Asthis order of events may be incidental, it cannotserve to deduce a direction of gene segment trans-fer. Indeed, the creation of a CVV-type virus byreassortment of a NORV M-segment into a POTVbackground cannot be excluded. POTV, NORV,MDV, and CVV are likely to overlap in geographicdistribution in central=northwestern states [3, 12,16, 33, 37]. Furthermore, POTV and CVV can both
2244 T. Briese et al.
replicate in Ae. albopictus mosquitoes [39, 38], andshare deer as an amplifying host reservoir [5, 37,40]. In addition, an overlap in KRIV and CVV ge-ography and host species is reported outside theUnited States. KRIV and CVV have been found inhorses in Argentina [13, 15]; viruses serologicallysimilar to CVV (BeAr 7272=MAGV) and KRIV(BeAr 8226) have been detected in Belem, Brazil[17, 19], and on the island of Trinidad (TR20659and TRVL8900=KRIV, respectively [1, 2, 21]). Thus,there has been ample opportunity for mixed infec-tions and spread of CVV=KRIV reassortant virusesinto ecologic niches in the northern hemisphere.The degree of identity observed between MDV
and CVV for S- and L-segment sequences (82% S-segment, nt level; 76% L-segment, nt level; 90%N, 94% NSs, and 85% L-protein, aa level), andbetween MDV and KRIV for M-segment sequence(70% at nt, and 73% at aa level), is lower than thatdetermined for the recently identified BUNV=BATV (NRIV) reassortment [10, 27]. This findingis compatible with a longer evolutionary historyof the MDV reassortment, resulting in increasedsequence divergence owing to genetic drift. Alter-natively, the greater divergence of MDV M-segmentsequence with respect to KRIV, in comparison tothat of MDV S- and L-segment sequences with re-spect to CVV or its subtype MAGV, may indicatethat KRIV is only a relative of an extinct or still-to-be-identified ultimate reassortment partner. Thisinterpretation is also in line with the fact thatKRIV, first isolated from mosquitoes collected in1955 in the Melajo forest on the island of Trinidad[2], has not been reported in North America. Anunrecognized direct M-segment ancestor may alsoapply in case of POTV. The M-segment sequenceidentity between POTV and KRIV (70% at nt and77% at aa level) is similar to that observed betweenMDV and KRIV. However, the closer match ofPOTV S-, and L-segment sequences to those ofCVV (89% S-segment, nt level; 82% L-segment,nt level; 99% N, 100% NSs, and 91% L-protein;aa level) points to a comparatively shorter indepen-dent history for POTV and CVV than for MDV andCVV, or MAGV.The divergent phylogenetic relationships ob-
served between S- and L-segment sequences versus
M-segment sequence suggest that POTV and MDVrepresent reassortants between CVV, or one of itsclose relatives, and virus(es) related to KRIV. Giv-en that the International Committee on Taxonomyof Viruses currently considers the species MainDrain virus and Kairi virus separate from theCache Valley virus, Potosi virus, and Northway vi-rus strains (or isolates) of Bunyamwera virus, ourfindings pose the question whether genome reas-sortment can occur between members of differentorthobunyavirus species, or alternatively, whetherthe capacity for genome reassortment can serve asa criterion to differentiate orthobunyavirus species.The accumulating sequence information for ortho-bunyaviral genome segments is beginning to revealdistinct genotypes that should allow dissection ofthe phylogenetic relationships for individual ge-nome segments and help to characterize the com-plex dynamics of orthobunyavirus evolution.
Acknowledgements
We thank Robert Tesh and the late Robert Shope of theUniversity of Texas Medical Branch, Galveston, and RobertLanciotti of the Centers for Disease Control and Prevention,Fort Collins, for providing virus stocks, and Cinnia Huangfor critical comments on the manuscript. This work wassupported by awards from the Ellison Medical Foundationand NIH (AI062705, AI056118, AI051292) to TB and WIL.
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