Natural M-Segment Reassortment in Potosi and Main Drain Viruses: Implications for the Evolution of Orthobunyaviruses

Home , Maguari orthobunyavirus, Robert Shope

Arch Virol (2007) 152: 2237–2247 DOI 10.1007/s00705-007-1069-z Printed 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 cpmc.columbia.edu [51]). They are unified by a Recently, we identified Batai virus as the M-segment common morphology that features a segmented ri- reassortment partner of Ngari virus. Extension of bonucleoprotein engulfed by a lipid envelope that is genetic analyses to other orthobunyaviruses related decorated with small glycoprotein spikes, forming to the Bunyamwera serogroup indicates additional mainly spherical particles of 80–120 nm; nonethe- natural genome reassortments. Whereas the relative less, the family is heterogeneous with respect to phylogenetic positions of all three genome segment host range and transmission mode. Whereas tospo- sequences were similar for Northway and Kairi vi- viruses infect plants, members of the other four ruses, the relative positions of Potosi and Main genera are animal viruses [51]. In addition, while Drain virus M-segment sequences diverged from the family encompasses the majority of described those of their S- and L-segments. Our findings in- arthropod-borne viruses (arboviruses) transmitted dicate M-segment reassortment in Potosi and Main by vectors such as thrips (e.g., tospoviruses), ticks Drain viruses and demonstrate natural genome (e.g., nairoviruses, phleboviruses), biting flies (e.g., reassortment as a driving force in the evolution of phleboviruses, nairoviruses) and mosquitoes (e.g., viruses of the Bunyamwera serogroup. orthobunyaviruses, phleboviruses), the members of the genus Hantavirus are not known to infect arthropods and are maintained in rodent reservoirs, Introduction adapted to a particular host species [42]. Although aspects of genome organization vary The Bunyaviridae are among the largest viral fami- among genera, a distinctive family feature is the lies, with over 300 members classified in the five presence of a tripartite single-stranded RNA ge- genera Orthobunyavirus, Nairovirus, Phlebovirus, nome that encodes replicase functions by the large Hantavirus, and Tospovirus (ICTVdb http:==phene. segment (L-segment), two surface glycoproteins by the medium size segment (M-segment), and a nucle- ocapsid protein (N) by the small genome segment Correspondence: Thomas Briese, Center for Infection and Immunity, 1801 Mailman School of Public Health, (S-segment) [51]. Among orthobunyaviruses, the Columbia University, New York, NY 10032, USA M-segment codes for a cotranslationally processed e-mail: [email protected] polyprotein that comprises the N- and the C-ter- 2238 T. Briese et al. minal glycoproteins, GN and GC, separated by a highly conserved domains were chosen according to com- small nonstructural membrane protein, NSm [23]. mon standards including avoidance of stable stem-loops, primer-dimer formation, and strong 30-terminal hybridiza- According to recent findings, NSm participates in tion. Sequence regions devoid of stable secondary structure virus assembly [52]. Another nonstructural protein, were sought for primer selection, however, compromises for NSs, coded by a second open reading frame (ORF) either one of these attributes had to be made for individual of the orthobunyaviral S-segment, has been shown permutations of degenerate primers. We recently described to counteract the innate host immune response by Greene SCPrimer, a software tool that in part integrates de- blocking alpha=beta interferon induction [7, 53]. sign constraints for selecting degenerate primers from multiple sequence alignments (http:==scprimer.cpmc.columbia. The segmented genome structure affords these edu [30]). viruses an opportunity for genome segment reassortment during a mixed infection event. Although ge- Virus isolates and nucleic acid extraction nome reassortment between viruses of the California Stocks of Potosi virus (POTV) strain 89–3380, Northway encephalitis (CE) or the Bunyamwera (BUN) sero- virus (NORV) strain 0234, Main Drain virus (MDV) strain group occurs readily in experimental settings [4, 6, BFS5015, and Kairi virus (KRIV) strain TRVL8900 were 26, 29, 48], natural reassortants are only infre- kindly provided by Robert Lanciotti, Robert Tesh, and the quently reported [27, 32]. Genetic analyses recently late Robert Shope. Eighty microliters of virus stock was extracted with Tri-Reagent following the manufacturer’s pro- identified UgMP-6830 from Uganda as an isolate tocol (MRC, Cincinnati, OH). Total RNA was dissolved in of Batai virus (BATV) and indicated that its M- 20 ml RNase-free H2O. segment most closely matched that of Ngari virus (NRIV), suggesting a historical M-segment reas- Reverse transcription – polymerase chain reaction sortment event [10]. Here, we present and analyze (RT-PCR) and sequencing S-, M-, and L-segment sequences of four other ortho- Three-microliter aliquots of total RNA were reverse tran- bunyaviruses: Potosi virus (POTV) and Northway scribed with random hexamers (Amersham Pharmacia virus (NORV), two North American viruses vec- Biotech, Uppsala, Sweden) in a 20-ml volume by using the Superscript II system (Invitrogen, Carlsbad, CA). PCR am- tored chiefly through Aedes mosquitoes within their plification [49] with various primer pairs was performed by deer or rodent reservoir, respectively; Main Drain incubating 0.5 ml cDNA, dNTP (200 mM), MgCl2 (3.0 mM), virus (MDV), vectored by Culicoides midges, but primers (1.6 mM, each), and Bio-X-act polymerase (1.6 units) also mosquitoes of the Aedes and Culiseta genera, in 25 ml 1 Opti buffer (supplied with polymerase; Bioline, Â between North American rodent reservoir hosts; London, UK) for 45 cycles in a PTC-200 thermocycler and Kairi virus (KRIV), vectored predominantly (MJ Research, Waltham, MA), applying a cycling protocol of 92 C for 1 min, 47 C, 48 C, or 52 C for 1 min (see by Aedes and Wyeomyia mosquitoes in largely un- Table 1), and 68 C for 1 min, followed by a final extension defined reservoirs throughout South and Middle for 10 min at 68 C. Amplification products were size-frac- America. All four viruses have been subsumed in tionated in 1.3% agarose gels and visualized by ethidium the Bunyamwera serogroup, although KRIV and bromide staining at the end of the run. Products were eluted MDV are serologically more distantly related to from gel fragments and sequenced either directly, resulting in a majority sequence, or where necessary after clon- other members of the group and are considered to ing into pGEM-Teasy plasmid vector (Promega, Madison, form separate serocomplexes [12]. Phylogenetic WI). Cloned sequences were obtained from at least three analysis of sequences from all three genome seg- plasmids for both strands by automated dideoxy-sequenc- ments suggests that POTV and MDV represent nat- ing [50] using BigDye Terminator Cycle Sequencing kits ural reassortment events. on ABI Prism Genetic Analyzer systems (Applied Bio- sytems, Foster City, CA). Sequence data generated by this work are available at Materials and methods GenBank under accession numbers AY729652, and EU004186-EU004194. Bioinformatics and primer design Full-length M-segment sequences for orthobunyaviruses of Sequence analyses and phylogenetic analysis the CE and BUN serogroups and Guaroa virus were retrieved Programs of the Wisconsin GCG Package (Accelrys, San from GenBank and analyzed for conservation. Primers in Diego, CA) were used for sequence assembly and analysis; Genetic analyses identify Potosi and Main Drain viruses as reassortant viruses 2239

Table 1. PCR ampliﬁcation primers

a Primer Sequence 30-position Annealing temperature S BUNS-5-U-6 50-CGGCGCC AGT AGT GTA CTC CAC 947 48 C BUNS-3-L947 50-GCGGCC AGT AGT GTG CTC CAC 15 Cac-2-FWD 50-dCT TAA CyT TGG rGG CTG GA 290 52 C Cac-7-REV 50-CTv ACr TTd Gyy TTC TTC CA 722 M BUN-S5-F 50-GCCGC AGT AGT GTA CTA CCG ATA yA 20 48 C M940C-R 50-CTr GCw GCT CTw AGr CTT TTr TAm CC 936 BUN-M-IXf 50-TGG GGn yGy GAr GAr Twy GG 3247 47 C BUN-M-XIIr 50-TTk GTy TTT TGk ACA TTk CC 3543 M3560-F 50-TCn AAr GGh TGy GGn AAT GT 3550 47 C BUN-S3-R 50-CGCGCC AGT AGT GTG CTA CC 4445 L BUNL-5n 50-CGCCGC AGT AGT GTA CTy CTA 15 48 C BUNL650R 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. percent sequence identities were calculated using ‘gap’ at BUN serogroups (Table 1 and Ref. [8]). Nucleotide default settings. Topology and targeting predictions were sequence was determined directly from amplifica- obtained by employing SignalP-NN SignalP-HMM, NetN- = tion products if permitted by size and quantity, or Glyc, TMHMM (http:==www.cbs.dtu.dk=services), the web- based version of TopPred2 (http:==bioweb.pasteur.fr= after cloning into a plasmid vector. To test the va- seqanal=interfaces=toppred.html), and Phobius (http:== lidity of initial draft sequences and to generate se- phobius.cgb.ki.se=index.html) [20, 31, 34, 43, 44]. Phyloge- quence for intervening regions not covered by netic analyses were performed by using MEGA 3.1 software products obtained with consensus primers, we used [35]. draft sequence to design POTV sequence-specific primers (primer sequences available upon request). The assembled M-segment consensus indicates Results a 1438-amino-acid (aa) coding sequence for the POTV polyprotein that shows over its entire length Acquisition of POTV sequence considerable divergence from the CVV M-segment Although POTV is reported to be indistinguishable sequence (60% nucleotide (nt) sequence and 56% from Cache Valley virus (CVV) in a cell-lysate an- aa sequence identity; Fig. 1). tigen enzyme-linked immunosorbent assay (ELISA) Interestingly, the use of amplification primers [3], it is readily differentiated from CVV in cross- outside of nt positions 1079–2169 of the POTV neutralization tests. The latter finding is in line with M-segment sequence yielded shorter products than the divergence of a POTV GN sequence fragment expected; products of only the expected size were from that of CVV [3, 27]. In an attempt to identify obtained when one primer was contained within the genetic basis for the observed reaction pattern, this region. Similar results were not observed with we determined and analyzed sequences from all NORV, MDV or KRIV (see ‘Comparative sequence three POTV genome segments. analyses’, below). Analysis of the amplification POTV sequence was amplified by RT-PCR using products was compatible with the presence of a consensus primers that target highly conserved do- deletion between nt 1079 and 2169, corresponding mains in the genomes of viruses of the CE and to aa positions 340–704 (Fig. 1). This observation is 2240 T . Briese Fig. 1. Multiple sequence alignment of M-segment sequences from viruses related to the BUN serogroup of orthobunyaviruses. NXT=S potential N-glycosylation site (grey shading); ___ potential transmembrane regions for predicted signal peptide (S) or membrane anchor (A); xxx conserved proteolytic cleavage motif et at C-terminus of G ; ttt conserved potential trypsin cleavage site [24]; - - - - - proposed fusion peptide domain [46]; conserved cysteine residue; deletion N Ã al. identified for POTV between nt 1079 (NSm) and 2169 (GC); Cons consensus sequence Genetic Table 2. S-Segment sequence conservation between POTV and other orthobunyaviruses

% Amino acid identityb % Nucleotide identityb analyses 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 identify POTV 98.7 86.8 83.6 83.3 82.0 83.0 81.1 80.6 79.1 79.6 71.3 67.4 63.9 (100)

NORV 96.2 94.9 86.3 84.2 80.8 84.4 80.2 79.2 79.8 80.2 73.2 67.0 63.4 Potosi (96.1) (96.1) MAGV 95.7 95.3 94.9 82.4 81.3 81.8 81.4 79.1 79.7 79.3 69.8 66.0 65.0 (97.1) (97.1) (95.1) and

MDV 89.7 89.3 91.9 91.5 78.3 80.1 79.6 78.4 76.4 76.0 69.3 68.0 63.6 Main (94.1) (94.1) (95.1) (93.1) BATV 94.0 93.6 94.9 93.2 89.3 86.6 78.0 78.9 76.4 76.9 72.3 63.8 62.7 Drain (94.1) (94.1) (94.1) (95.1) (90.2) AUSa 95.3 94.9 95.3 92.7 88.9 97.4 80.0 79.1 78.4 78.2 72.0 68.8 61.1 (98.0) (98.0) (94.1) (95.1) (92.2) (94.1) viruses BUNV 90.6 91.0 92.7 91.0 90.2 91.9 92.7 95.1 85.0 85.1 72.9 68.1 61.8 (88.2) (88.2) (88.2) (88.2) (86.3) (88.2) (86.3) as NRIV 89.3 89.7 92.8 91.0 89.2 92.4 92.8 99.6 85.5 85.5 70.2 67.8 62.4 reassortant (88.0) (88.0) (89.1) (88.0) (87.0) (89.1) (85.9) (100) ILEV 90.2 89.7 90.2 88.5 86.8 89.3 90.6 94.0 93.3 98.0 69.1 66.7 63.0 (87.3) (87.3) (85.3) (85.3) (85.3) (85.3) (85.3) (95.1) (94.6) MBOV 90.2 89.7 90.2 88.5 86.8 89.7 90.6 94.0 93.7 99.2 69.1 66.7 61.4 viruses (88.2) (88.2) (86.3) (86.3) (84.3) (86.3) (86.3) (94.1) (93.5) (99.0) GERV 75.2 74.4 76.1 73.9 74.4 77.4 76.9 75.2 74.9 74.8 75.2 62.8 58.3 (70.3) (70.3) (67.3) (68.3) (66.3) (67.3) (69.3) (70.3) (70.3) (70.3) (70.3) KRIV 69.2 69.2 69.7 68.0 70.5 68.4 69.2 70.9 71.3 71.8 71.8 62.4 64.7 (68.6) (68.6) (69.6) (67.7) (68.3) (66.7) (67.7) (66.7) (67.4) (65.7) (65.7) (56.0) GROV 70.9 70.9 70.1 69.2 68.8 70.5 70.5 68.8 69.5 68.0 68.4 63.7 69.7 (42.9) (42.9) (44.1) (41.7) (46.4) (42.9) (42.9) (41.7) (44.6) (44.1) (42.9) (41.7) (50.0) a Uncharacterized Australian Bunyamwera virus isolate AF325122. b Nucleotide conservation is indicated in the upper right half of the table. Amino acid conservation is indicated in the lower left half of the table; N sequences, plain text; NSs sequences, text in parentheses. 2241 2242 T. Briese et al. reminiscent of a finding in Maguari virus (MAGV) peptide sequences (Fig. 1). This structure is consis- revertants, where similar deletions spanning the tent with earlier bioinformatics predictions based NSm and N-terminal GC region were generated on Guaroa virus (GROV) M-segment sequence [8] during reversion from a temperature-sensitive mu- and experimental data recently obtained for the tant phenotype [47]. The presence of NSm-deleted NSm of Bunyamwera virus (BUNV) [52]. The ex- as well as sequences co-linear to other ortho- tended set of available sequences indicates conser- bunyaviral genome sequences reveals a notable vation of only three potential N-glycosylation sites: genomic sequence heterogeneity of POTV stock an N- and a C-terminal site in GN, and a single site virus. located C-terminally in GC (Fig. 1). Other potential The S-segment sequence of POTV was similar glycosylation sites appear to follow more type-spe- in length to other BUN S-segment sequences [22], cific patterns. Sequence conservation for GN is encoding an N protein of 233 aa and an NSs protein higher than for NSm or GC and is most pronounced of 101 aa that is translated from an ORF overlap- in a region between the conserved C-terminal gly- ping in the 1 frame with that of N. Remarkably, cosylation site and the terminal protease cleavage the POTV Sþ-segment sequence shares a high de- motif KSLRV=AAR [24]. The topological model gree of sequence identity with that of CVV at the for NSm predicts that this region has a cytoplasmic nt level (89%) as well as at the aa level for both location. Conservation is also noted for a motif the N and the NSs ORFs (99% and 100%, respec- in the cytoplasmic domain of NSm, and for three tively) (Table 2). C-terminal motifs located around the conserved Last, we amplified a portion of the POTV L- glycosylation motif of GC (Fig. 1), including a pro- segment. The sequence had less than 74% se- posed fusion peptide domain [46]. Highest sequence quence identity at the nt level (81% at the aa level) variability is found in an approximately 80-aa region to BATV, the most closely related orthobunyavirus preceding the conserved potential trypsin recogni- L-segment sequence available in GenBank at the tion site in GC [24]. time of this analysis (data not shown). In an effort to identify sequences closely related Phylogenetic analysis to the M- and L-segment sequences determined for POTV, we applied our panel of consensus primers The reconstructed phylogeny based on S-segment to PCR amplification of corresponding sequences nt sequence identifies POTV, together with NORV, from NORV, MDV and KRIV, using the same strat- as the closest matches to the CVV sequence. The egy as outlined for POTV. overall topology was consistent, irrespective of the analysis model applied; Fig. 2A shows a consensus tree of 1000 bootstrap repetitions applying a Comparative sequence analyses Tamura-Nei Neighbor-Joining model. Integrating Analysis of POTV, NORV, MDV and KRIV M-seg- recently determined S-segment sequences did not ment sequence indicated a highly divergent length significantly change the tree topology compared to and sequence of their untranslated regions (UTRs) that reported by Dunn et al. [22]. However, the outside of the conserved segment termini. Close additional sequences did indicate distinct genetic sequence homology was observed only between clades within the serogroup. A CVV-related clade the UTRs of CVV and MAGV (data not shown), includes CVV, POTV, NORV, MAGV, and MDV, as and those of BATV and NRIV [10]. The coding well as more distantly related the BATV sequences. sequences showed the common orthobunyavirus A BUNV-related clade encompasses the African organization with mature proteins in the order GN- viruses BUNV=NRIV, Ilesha (ILEV) and Mboke NSm-GC. Topological analyses of POTV, NORV, (MBOV), which separate from the phylogenetically MDV, and KRIV coding sequence indicate that more distant Germiston virus (GERV), KRIV, and NSm has a luminal N-, and a cytoplasmic C-termi- GROV sequences (Fig. 2A). Although relationships nal domain framed by two potential internal signal for N and NSs aa sequences are similar to those Genetic analyses identify Potosi and Main Drain viruses as reassortant

Fig. 2. Phylogenetic analysis of selected orthobunyavirus S-, M-, and L-segment sequences. Phylogenetic trees based on (A) S-, (B) M-, and (C) partial L-

segment nucleotide sequence (approx. 570 nt) were reconstructed by the Neighbor-Joining method applying a Tamura-Nei model with 1000 pseudoreplicates; viruses boostrap values above 60% are shown at the respective branches. GenBank accession numbers for the respective sequences are indicated. California serogroup viruses Serra do Navio (SDNV), La Crosse (LACV), and prototype California encephalitis (CEV), as well as Simbu serogroup virus Oropouche (OROV), are included for comparison. Shading indicates viruses analyzed in this study 2243 2244 T. Briese et al. observed with nt sequence, the MDV aa sequences of the M-segments to generate the second of these are placed by some software models in a closer re- two reassortant viruses. lationship to Asian and African sequences (data not POTV was first isolated in 1989 when Aedes shown [22]). albopictus mosquitoes from Potosi, Missouri, The overall topology of phylogenetic trees ob- were investigated for arbovirus infection [25]. Ae. tained with M-segment sequences and S-segment albopictus mosquitoes, native to Asia, had been sequences is similar; however, POTV and MDV detected in 1985 in Houston, Texas, and the appear- are more closely related to KRIV than to CVV in ance of Ae. albopictus larvae the subsequent year their M-segment sequence (Fig. 2A, B). Interestingly, in imported tire casings at the port of Seattle, although MAGV is considered to be a subtype of Washington, suggested a potential route for intro- CVV [14], POTV and CVV are closer in S-segment duction of this new vector to the United States. sequence than are CVV and MAGV. Compared to However, lack of vertical transmission of POTV that, POTV and KRIV appear to be less similar in by Ae. albopictus mosquitoes argued against an in- their M-segment sequence. Phylogenetic analysis troduction of the new virus into the Potosi area of M-segment sequences indicates a closer relation- through infected eggs [38]; furthermore, cross-neu- ship between GERV and BUNV, and between ILEV tralization tests did not indicate a relationship of and the CVV clade than indicated by the S-segment POTV to Asian BATV. Indeed, the original report sequence analysis. of POTV describes an inefficient neutralization by Given the highly divergent relationships ob- KRIV antibodies [25], a result in line with the se- served for the S- and M-segment sequences of quence findings reported here. POTV and MDV, it was of interest to assess how Molecular studies indicate that POTV has fre- their L-segment sequences would match with that quently been misidentified in ELISA as CVV, or of CVV or KRIV. Thus, we obtained L-segment se- even Jamestown Canyon virus [3], and the develop- quence of CVV strain 6V633, as well as of MAGV ment of specific detection assays suggest that strain BeAr7272, for a region corresponding to POTV has a much wider distribution than previous- L-segment sequences reported for other BUN ser- ly anticipated [3, 41]. These findings and our own ogroup viruses. Although the absence of a large emphasize the complementing role of molecular sequence repertoire precludes phylogenetic analy- diagnostics and highlight the importance of im- sis as comprehensive as that achieved for S and plementing broad-range diagnostic tools such as M segments, the POTV and MDV L-segment se- multiplex PCR or microarray platforms that are quences appear more closely related to CVV, capable of considering not only one suspected path- MAGV and NORV than to KIRV (Fig. 2C). ogen, but interrogate a wide range of potential can- didates simultaneously [9, 36, 45]. Since the isolation of CVV in 1956 [28], other Discussion newly discovered orthobunyaviruses considered to Phylogenetic analysis of sequences from all three be native to North America include MDV, isolated genome segments of POTV, NORV, MDV, CVV, from midges collected in California in 1964 [11], MAGV and KRIV indicates a divergent phylogenet- and NORV, identified in mosquitoes and sentinel ic relationship for POTV and MDV M-segment rabbits at Northway, Alaska, in 1970=71 [18]. As sequences in comparison to that of their S- and this order of events may be incidental, it cannot L-segment sequences. In contrast, consistent rela- serve to deduce a direction of gene segment trans- tive phylogenetic relationships are observed for all fer. Indeed, the creation of a CVV-type virus by three genome segment sequences of NORV and reassortment of a NORV M-segment into a POTV KRIV, as well as CVV and MAGV. These findings background cannot be excluded. POTV, NORV, suggest at least one M-segment reassortment event MDV, and CVV are likely to overlap in geographic in the evolutionary history of POTV and MDV, in- distribution in central=northwestern states [3, 12, cluding possibly a secondary reassortment of one 16, 33, 37]. Furthermore, POTV and CVV can both Genetic analyses identify Potosi and Main Drain viruses as reassortant viruses 2245 replicate in Ae. albopictus mosquitoes [39, 38], and M-segment sequence suggest that POTV and MDV share deer as an amplifying host reservoir [5, 37, represent reassortants between CVV, or one of its 40]. In addition, an overlap in KRIV and CVV ge- close relatives, and virus(es) related to KRIV. Giv- ography and host species is reported outside the en that the International Committee on Taxonomy United States. KRIV and CVV have been found in of Viruses currently considers the species Main horses in Argentina [13, 15]; viruses serologically Drain virus and Kairi virus separate from the similar to CVV (BeAr 7272=MAGV) and KRIV Cache Valley virus, Potosi virus, and Northway vi- (BeAr 8226) have been detected in Belem, Brazil rus strains (or isolates) of Bunyamwera virus, our [17, 19], and on the island of Trinidad (TR20659 findings pose the question whether genome reas- and TRVL8900=KRIV, respectively [1, 2, 21]). Thus, sortment can occur between members of different there has been ample opportunity for mixed infec- orthobunyavirus species, or alternatively, whether tions and spread of CVV=KRIV reassortant viruses the capacity for genome reassortment can serve as into ecologic niches in the northern hemisphere. a criterion to differentiate orthobunyavirus species. The degree of identity observed between MDV The accumulating sequence information for ortho- and CVV for S- and L-segment sequences (82% S- bunyaviral genome segments is beginning to reveal segment, nt level; 76% L-segment, nt level; 90% distinct genotypes that should allow dissection of N, 94% NSs, and 85% L-protein, aa level), and the phylogenetic relationships for individual ge- between MDV and KRIV for M-segment sequence nome segments and help to characterize the com- (70% at nt, and 73% at aa level), is lower than that plex dynamics of orthobunyavirus evolution. determined for the recently identified BUNV= BATV (NRIV) reassortment [10, 27]. This finding Acknowledgements is compatible with a longer evolutionary history of the MDV reassortment, resulting in increased We thank Robert Tesh and the late Robert Shope of the sequence divergence owing to genetic drift. Alter- University of Texas Medical Branch, Galveston, and Robert natively, the greater divergence of MDV M-segment Lanciotti of the Centers for Disease Control and Prevention, Fort Collins, for providing virus stocks, and Cinnia Huang sequence with respect to KRIV, in comparison to for critical comments on the manuscript. This work was that of MDV S- and L-segment sequences with re- supported by awards from the Ellison Medical Foundation spect to CVV or its subtype MAGV, may indicate and NIH (AI062705, AI056118, AI051292) to TB and WIL. that KRIV is only a relative of an extinct or still- to-be-identified ultimate reassortment partner. This References interpretation is also in line with the fact that KRIV, first isolated from mosquitoes collected in 1. Aitken TH, Spence L (1963) Virus transmission studies 1955 in the Melajo forest on the island of Trinidad with Trinidadian mosquitoes. III. Cache Valley virus. West Indian Med J 12: 128–132 [2], has not been reported in North America. 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