Natural M-Segment Reassortment in Potosi and Main Drain Viruses: Implications for the Evolution of Orthobunyaviruses
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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 mul- tiple sequence alignments (http:==scprimer.cpmc.columbia. The segmented genome structure affords these edu [30]). viruses an opportunity for genome segment reassort- ment 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, result- ing 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.