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Complete Coding Sequence of the Alkhurma Virus, a Tick-Borne Flavivirus Causing Severe Hemorrhagic Fever in Humans in Saudi Arabia

Complete Coding Sequence of the Alkhurma Virus, a Tick-Borne Flavivirus Causing Severe Hemorrhagic Fever in Humans in Saudi Arabia
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  Complete Coding Sequence of the Alkhurma Virus,a Tick-Borne Flavivirus Causing Severe HemorrhagicFever in Humans in Saudi Arabia Re´mi N. Charrel,* , † ,1  Ali Mohamed Zaki,‡ Houssam Attoui,* Mazen Fakeeh,‡ Fre´de´rique Billoir,* Amany Ibrahim Yousef,‡ Reine de Chesse,* Philippe De Micco,* Ernest A. Gould,§and Xavier de Lamballerie* , † * Unite´ des Virus Emergents EA 3292, Universite´ de la Me´diterrane´e, Faculte´ de Me´decine, 27 boulevard Jean Moulin, Marseille 13005, France;  †  Maladies virales e´mergentes & syste`mes d’information UR 034, Institut de Recherche pour le De´veloppement, France;  ‡ Virology Laboratory, Dr. Suliman Fakeeh Hospital, P.O. Box 2537, Jeddah, Saudi Arabia; and §  National Environment Research Council Institute of Virology and Environmental Microbiology, Oxford, United Kingdom Received August 17, 2001 To date, tick-borne flaviviruses responsible for hem-orrhagicfeverinhumanshavebeenisolatedinSiberia(Omsk hemorrhagic fever virus), India (KyasanurForest disease virus, KFDV), and in Saudi Arabia(Alkhurma virus, ALKV). Prior to this study, only par-tial coding sequences of these severe pathogens hadbeen determined. We report here the complete coding sequence of ALK virus, which was determined to be10,248 nucleotides (nt) long, and to encode a single3,416 amino acid polyprotein. Independent analyses of the complete polyprotein and the envelope proteinprovided genetic and phylogenetic evidence that ALKV belongs to the tick-borne flavivirus group,within which it is most closely related to KFDV. Anal-ysis of structural genes, genetic distances, and evolu-tionary relationship indicate that ALKV and KFDV derived from a common phylogenetic ancestor andconstitute two genetic subtypes of the same virus spe-cies according to current genetic criteria of classi-fication.  © 2001 Academic Press  Key Words:  flavivirus; Flaviviridae; phylogeny; tick-borne virus; hemorrhagic fever; Alkhurma virus. In 1995, a virus tentatively named Alkhurma virus(ALKV) and related to the tick-borne (TB) flaviviruses(1) was isolated from the blood of several patients withsevere hemorrhagic fever in Saudi Arabia. Since thattime, a total of 16 cases has been confirmed by virusisolation, of which 4 had a fatal outcome. The discoveryof this virus was considered to be an important eventbecause TB flaviviruses responsible for hemorrhagicfever in humans had been isolated previously only inSiberia (Omsk hemorrhagic fever virus, OHFV) and inIndia (Kyasanur Forest disease virus, KFDV). The se-quence determination of ALKV NS5 gene suggestedthat the virus was closely related to KFDV (1), one of the most pathogenic TB flaviviruses, causing hemor-rhagic manifestations with a case fatality rate of 2 to10%(2).Itwasfirstrecognizedin1957intheKyasanurForest (Shimoga District, India) (3) and causes annu-ally an average of 500 cases. However, genetic compar-ison of ALKV with TB flaviviruses (an in particularwith OHFV) was hampered by the lack of genetic in-formation in the structural genes, which have beenmostly studied in previous studies of TB-flaviviruses.In this study, we report the sequence determinationof the complete open reading frame (ORF) of the ALKV prototype strain. This constitutes the first completegenetic characterization of a TB-complex flavivirus re-sponsible for hemorrhagic manifestations. Compara-tive analyses with other mammalian TB flavivirusesand phylogenetic studies are presented and discussed. MATERIALS AND METHODS Viral strain.  The ALKV prototype strain 1176 was recovered in1995 from the blood of a patient with fever, headache, retro-orbitalpain, joint pain, generalized muscle pains, anorexia, and vomiting;the strain was successively passaged twice in suckling mouse brains,three times in Vero cells, once in sheep and finally once again innewborn mice. The current study was performed on viral RNA ex-tracted from these newborn mice brains.  RNA extraction.  RNA was extracted using the RNA NOW TC-Kit(Biogentex. Inc., Seabrook, TX) according to the manufacturer’s in-structions, resuspended into 50   l of RNase-free sterile water, andstored at  70°C until processed. 1 To whom correspondence should be addressed at Unite´ des VirusEmergents, Faculte´ de Me´decine, 27, boulevard Jean Moulin, Mar-seille 13005, France. Fax: (33) 491 32 44 95. E-mail: rnc-virophdm@gulliver.fr.Biochemical and Biophysical Research Communications  287,  455–461 (2001)doi:10.1006/bbrc.2001.5610, available online at http://www.idealibrary.com on455 0006-291X/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.   Reverse transcription.  RT was carried out at 42°C in a 20-  lreaction mixture that included 11   l of RNA extract, 200 U of Superscript II RNase H  Reverse Transcriptase (Life Technologies,Inc., Grand Island, NY) and 2 pmol of oligonucleotide 3PNC-2R(5  -GCTCAGGGAGAACAAGAACCG-3  ) located in the 3  noncoding region. The reaction mixture was subsequently treated with DNase-free RNase (Roche Diagnostics, Meylan, France). The resulting non-infectious subgenomic cDNA was received at the Unite´ des VirusEmergents and further processed.  Polymerase chain amplification and sequencing reactions.  DNA products were amplified by PCR from 10 overlapping regions (Fig. 1).PCRs were carried out in a volume of 50   l that included 10 mMTris–HCl [pH 9.0], 1.5 mM MgCl2, 50 mM KCl, 0.1% Triton X-100,200   M each dNTP, 0.2   M of each primer, 5   l of cDNA and 1.5 Uof   Taq  DNA polymerase (Promega Corp., Madison, WI). The thermo-cycler profile was 5 min at 95°C, followed by 35 cycles of 30 s at 95°C,1 min at 50°C, and 2 min at 72°C, with a 7-min final extension at72°C. PCR products were purified from agarose gel (Wizard PCRPreps DNA Purification System, Promega Corp.) and directly se-quenced using the ABI Prism Dye Terminator Cycle Sequencing Ready Reaction Kit (Perkin–Elmer Corp., Foster City, CA) and an ABI Prism 377XL automated sequencer (Perkin–Elmer Corp.). Sequence data and phylogenetic analysis.  Full-length coding se-quences of TB flaviviruses were retrieved from the GenBank database(Table 1) together with the following sequences: yellow fever virusstrain 17D (YFV, X03700); dengue 1 virus strain Western Pacific(DENV-1, M88535); dengue 2 virus strain New Guinea C (DENV-2, AF038403); dengue 3 virus strain H87 (DENV-3, M93130); dengue 4virus (DENV-4, M14931); Kunjin virus strain MRM61C (KUNV,D00246); Japanese encephalitis virus strain JaOArS982 (JEV,M18370), West Nile virus strain Eg101 (WNV, AF260968); St. Louisencephalitis virus strain MI-7 (SLEV, AF160194); Murray Valley en-cephalitis virus strain MVE-1-51 (MVEV, NC 000943); mosquito cellfusing agent (CFAV, M91671); Rio Bravo virus strain M-64 (RBV, AF144692), Apoi virus strain Kitaoka (APOIV, AF160193).These complete flavivirus amino acid (AA) sequences were alignedusing the Clustal W 1.7 program (4). This permitted (i) to deduce theputative cleavage sites of the ALKV polyprotein and (ii) to calculate AA sequence identities using the pairwise distance algorithm of theMEGA software program (5).Phylogenetic relationships were determined using these pairwisedistances and the neighbor-joining (NJ) method implemented inMEGA. The robustness of the resulting branching patterns wastested by bootstrap analysis with 500 replications and the correspon-dence of phylogenetic groupings with previously described serocom-plexes was analyzed. An additional analysis was performed using partial AA sequences intheenveloperegionretrievedfromtheGenBankdatabase(seeTable2). Alignments and phylogenetic analysis were performed as describedabove. The observed intra- and interspecies genetic distances wereanalyzed and used to discuss the taxonomic assignment of ALKV. RESULTS AND DISCUSSION The complete coding sequence of ALKV strain1176was obtained by sequencing 10 overlapping PCR am-plification products (Fig. 1).The coding sequence wasdetermined to be 10,248 nucleotides (nt) in length,including a single ORF that encodes a 3,416 amino acid(AA) polyprotein (GenBank Accession No. AF331718).Comparison with the complete AA sequences of otherflaviviruses clearly confirmed that ALKV is closely re-lated to mammalian TB flaviviruses. In particular, theanalysis of complete sequence data (Fig. 2) showed thatthe genetic distance between ALKV and non-TB flavi-viruses is higher or equal to 57%, whereas it rangedfrom 21 to 24.3% between ALKV and TB flaviviruses, avalue within the range observed between previouslycharacterized members of the TB group (  25.6%). Sim- TABLE 1 Mammalian Tick-Borne Flaviviruses Used in the Phylogenetic (Fig. 3B) Study and Taxonomic Study (Fig. 4)  Virus species a Subtypes and isolates Abbreviation a GenBank Accession No. Sequence Origin  Alkhurma virus  Human isolate ALKV AF331718 Complete Saudi Arabia  Kyasanur Forest disease virus  Human isolate KFDV X74111 Envelope India  Langat virus Ixodes granulatus  LGTV M73835 Complete Malaysia Omsk hemorrhagic fever virus Dermacentor marginatum  OHFV X66694 Envelope Siberia  Powassan virus  Human isolate POWV L06436 Complete Ontario  Deer-tick virus b Deer-tick isolate DTV AF135461 Envelope Wisconsin Tick-borne encephalitis virus  European subtype TBEV  HYPR U39392 Complete Eastern Europe Tick-borne encephalitis virus  European subtype TBEV  NEU U27495 Complete Eastern Europe Tick-borne encephalitis virus  Far Eastern subtype TBEV  SOF GNWVTB Complete c Far-East Russia Tick-borne encephalitis virus  Siberian subtype TBEV   VAS L40361 Complete Siberia  Louping ill virus  Irish subtype LIV  IR CAA60480 Envelope Ireland  Louping ill virus  British subtype LIV  BR D12937 Envelope UK   Louping ill virus  Spanish subtype LIV  SSE CAA54619 Envelope Spain  Louping ill virus  Turkish subtype LIV  TSE S41628 Envelope Turkey  Louping ill virus  Greek subtype LIV  GGE  X77732 Envelope Greece  Louping ill virus  Other isolates LIV  NEG M94956 Envelope Japan  Louping ill virus  Other isolates LIV  WAL CAA60481 Envelope UK (Wales)  Louping ill virus  Other isolates LIV  369  Y07863 Envelope UK   Louping ill virus  Other isolates LIV  NOR D12936 Envelope Norway a  As referred in the VIIth Report of the ICTV (11). b Proposed as a genotype of POWV by Telford  et al.  (12). c  Amino acid sequence available only. Vol. 287, No. 2, 2001 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS456  ilarly, the phylogenetic analysis performed with com-plete coding sequences (Fig. 3A) permitted to include ALKV in the TBE group with a 100% bootstrap value.Finally, the analysis of AA alignments permitted un-ambiguous identification of the putative cleavage sitesof ALKV polyprotein (Table 2A), by reference to previ-ously determined cleavage sites (6). The AnchC, PrM,M, and NS2A, NS2B, NS3, NS4A, 2K, NS4B and NS5proteins are identical in length in all the TB flavivi-ruses included in this study (Table 2B). [The NS4B andNS5 proteins of TBEV  SOF reported here are slightlydifferentin lengthfrom thoseofthesequencedepositedin databases (GNWVTB). They were deduced from acorrected partial sequence of this strain (kindly pro-vided by Dr. Pletnev and Dr. T. Gritsun, personaldata), and conform with those reported for all otherTBEV strains sequenced to date. Accordingly, allTBEV, LIV and LGTV strains have putative structuraland nonstructural proteins that are identical inlength.] The 3 genes for which differences were ob-served are VirC, E and NS1. The E protein is 496 AA long for all TB flaviviruses, including ALKV andKFDV; the only exception is POWV (497 AA). VirC is96 AA long in TBEV, LIV, LGTV, 94 AA long in POWV,and 97 AA long for both ALKV and KFDV. NS1 proteinis 353 AA long for TBEV, LIV and LGTV, and 354 AA long for ALKV and POWV (not determined for KFDV)(Table 2B). Therefore, ALKV possesses the longestpolyprotein of all TB-flaviviruses characterized so far.The relatedness of ALKV with TB flaviviruses wasalso demonstrated by the analysis of the E protein (7). A number of conserved patterns could be identifiedincluding: (i) the 12 cysteine residues involved in theintramolecular disulfide bonds; (ii) the 3 potentialN-glycosylation sites (positions 154–156, 361–363, and473–475); (iii) the TB-flavivirus-specific pentapeptideDSGHD previously described (8) at positions 320-324;(iv) the sequence of the ALKV fusion peptide (positions98–111) was identical to that of TBEV, LIV, LGTV,and KFDV, and 1 AA different from that of POWV. FIG. 1.  Strategy used for sequencing the genome of ALKV. The topmost diagram shows a typical flavivirus full-length coding sequence.The different genes and their respective size are indicated. Each graduation corresponds to 1000 nucleotides. Sequencing strategy of ALKV genome was based on the amplification of 10 overlapping PCR products (P1 to P10). Primers sequences are available upon simple requestto the corresponding author. FIG.2.  Matrix of genetic distances. The upper-right matrix represents pairwise distances between standard amino acids alignments. Thelower-left matrix represents pairwise distances between standard nucleotides alignments. Groupings at the top of the figure were made using 0.450 and 0.380 cutoff values for amino acids and nucleotides respectively. Correspondence with serocomplexes is indicated. Vol. 287, No. 2, 2001 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS457   Vol. 287, No. 2, 2001 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS458  Further analysis of AA motifs showed that ALKV ismore closely related to KFDV (7): (i) the KFDV specific AKG motif at positions 2–4 of the VirC protein is alsopresent in the ALKV polyprotein; (ii) the insertion of 1 AA residue at position 93 of the VirC protein is foundonly in ALKV and KFDV polyproteins; this residue isan arginine for both viruses; (iii) the KFDV specificEHLPKA hexapeptide at positions 207–212 of the Eprotein is present in the ALKV polyprotein (8); (iv) theKFDV specific EGSK motif (position 308–311 of the Eprotein) that was related to the non reactivity of Mab4.2 with KFDV E protein is identical in ALKV (9); (v) ALKV and KFDV share the same AA substitutions atpositions 76 and 489 of the E protein.However, it is possible to distinguish between ALKV and KFDV in the hypervariable region of the E protein(positions 232–234) (10) where ALKV encodes the se-quence AHE distinct from the AQE motif of KFDV (7).Therefore, whether ALKV represents a species distinctfrom KFDV or a new subtype within the KFDV speciesrequired further investigation.To address this question, phylogenetic analysis wasperformed within a 496 AA region of the envelope genewhere sequences are available for 19 TB flaviviruses.The topology of the tree obtained (Fig. 3B) is consistentwith that observed using complete ORF sequences butbrings the additional information that ALKV andKFDV derive from a common ancestor (assessed by a100% bootstrap value). AA distances were used tostudy the distribution of evolutionary distances uponpairwise comparison (Fig. 4A). The bimodal shape of the curve revealed a cutoff value at 14%, above which AA distances correspond to strains that belong to dis-tinct TB-virus species according to the ICTV classifi-cation (11). The only noticeable exception was repre-sented by LIV and TBEV strains that could not bedifferentiated using this cutoff value. The distanceranges within TBEV strains (up to 7.3%, Fig. 4B) orLIV strains (up to 4.8%, Fig. 4C), and between TBEV and LIV strains (3.8–9.3%, Fig. 4D) are widely over-lapping. There is therefore no evidence from the distri-bution of genetic distances that TBEV and LIV consti-tute distinct species. However, in the case of ALKV andKFDV, the observed 3.0% distance is compatible onlywith these viruses belonging to the same virus speciessuggesting that ALKV may be considered a geneticsubtype of KFDV.The ecological conditions that determine virus sur-vival and propagation are very different for KFDV inIndia (3) compared with ALKV in Saudi Arabia. Hu- FIG. 4.  Distribution of evolutionary distances upon pairwisecomparison. The distances were calculated from a 496-AA regionlocated in the E protein (position 1–496) The genetic distance isreported on the x-axis. Frequency of genetic distances is recorded onthe y-axis. (A) Distribution of the distances observed between the 19strains of tick-borne flaviviruses presented in Table 1. The shadedsquare represents AA distances (18.7–20.8%) observed between ALKV and LIV, TBEV, LGTV strains. The gray arrow corresponds tothe AA distance observed between ALKV and KFDV. The blackarrowrepresentsthecutoffforspeciesidentification.(B)Distributionof the distances observed within the TBEV strains. (C) Distributionof the distances observed within the LIV strains. (D) Distribution of the distances observed between TBEV and LIV strains. FIG. 3.  Phylogenetic analysis of flaviviruses based on complete polyprotein (A) and a 496-AA region of the envelope (B). Distances andgroupings were determined by the pairwise distance algorithm and the neighbor-joining method using the MEGA software program (5).Bootstrap values are indicated and correspond to 500 replications. In A, the European subtype TBEV strains group with LIV  369 , while theSiberian and Far Eastern subtype TBEV strains form a distinct subgroup. The ALKV branch is located between that of POWV and LGTV,as previously reported for KFDV from the phylogenetic analysis of envelope sequences (7). Vol. 287, No. 2, 2001 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS459
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