Genetic detection and characterization of Lujo virus, a new hemorrhagic fever-associated arenavirus from southern Africa

Abstract

Lujo virus (LUJV), a new member of the family Arenaviridae and the first hemorrhagic fever-associated arenavirus from the Old World discovered in three decades, was isolated in South Africa during an outbreak of human disease characterized by nosocomial transmission and an unprecedented high case fatality rate of 80% (4/5 cases). Unbiased pyrosequencing of RNA extracts from serum and tissues of outbreak victims enabled identification and detailed phylogenetic characterization within 72 hours of sample receipt. Full genome analyses of LUJV showed it to be unique and branching off the ancestral node of the Old World arenaviruses. The virus G1 glycoprotein sequence was highly diverse and almost equidistant from that of other Old World and New World arenaviruses, consistent with a potential distinctive receptor tropism. LUJV is a novel, genetically distinct, highly pathogenic arenavirus.

Figure 1. Geographic distribution of African arenaviruses.

MOBV, MOPV, and IPPYV (blue) have not been implicated in human disease; LASV (red) can cause hemorrhagic fever. The origin of the LUJV index and secondary and tertiary cases linked in the 2008 outbreak are indicated in gold.

Figure 2. LUJV genome organization and potential secondary structure of intergenic regions.

Open reading frames (ORF) for the glycoprotein precursor GPC, the nucleoprotein NP, the matrix protein analog Z, and the polymerase L, and their orientation are indicated (A); blue bars represent sequences obtained by pyrosequencing from clinical samples. Secondary structure predictions of intergenic regions (IR) for S (B, C) and L segment sequence (D, E) in genomic (B, D) and antigenomic orientation (C, E) were analyzed by mfold; shading indicates the respective termination codon (opal, position 1), and its reverse-complement, respectively.

Figure 3. Phylogenetic analyses of LUJV.

Phylogenetic relationships of LUJV were inferred based on full L (A) and S segment nucleotide sequence (B), as well as on deduced amino acid sequences of L (C), NP (D), Signal/G2 (E) and G1 (F) ORF's. Phylogenies were reconstructed by neighbor-joining analysis applying a Jukes-Cantor model; the scale bar indicates substitutions per site; robust boostrap support for the positioning of LUJV was obtained in all cases (>98% of 1000 pseudoreplicates). GenBank Accession numbers for reference sequences are: ALLV CLHP2472 (AY216502, AY012687); AMAV BeAn70563 (AF512834); BCNV AVA0070039 (AY924390, AY922491), A0060209 (AY216503); CATV AVA0400135 (DQ865244), AVA0400212 (DQ865245); CHPV 810419 (EU, 260464, EU260463); CPXV BeAn119303 (AY216519, AF512832); DANV 0710-2678 (EU136039, EU136038); FLEV BeAn293022 (EU627611, AF512831); GTOV INH-95551 (AY358024, AF485258), CVH-960101 (AY497548); IPPYV DakAnB188d (DQ328878, DQ328877); JUNV MC2 (AY216507, D10072), XJ13 (AY358022, AY358023), CbalV4454 (DQ272266); LASV LP (AF181853), 803213 (AF181854), Weller (AY628206), AV (AY179171, AF246121), Z148 (AY628204, AY628205), Josiah (U73034, J043204), NL (AY179172, AY179173); LATV MARU10924 (EU627612, AF485259); LCMV Armstrong (AY847351), ARM53b (M20869), WE (AF004519, M22138), Marseille12 (DQ286932, DQ286931), M1 (AB261991); MACV Carvallo (AY619642, AY619643), Chicava (AY624354, AY624355), Mallele (AY619644, AY619645), MARU222688 (AY922407), 9530537 (AY571959); MOBV ACAR3080MRC5P2 (DQ328876, AY342390); MOPV AN20410 (AY772169, AY772170), Mozambique (DQ328875, DQ328874); NAAV AVD1240007 (EU123329); OLVV 3229-1 (AY216514, U34248); PARV 12056 (EU627613, AF485261); PICV (K02734), MunchiqueCoAn4763 (EF529745, EF529744), AN3739 (AF427517); PIRV VAV-488 (AY216505, AF277659); SABV SPH114202 (AY358026, U41071); SKTV AVD1000090 (EU123328); TAMV W10777 (EU627614, AF512828); TCRV (J04340, M20304); WWAV AV9310135 (AY924395, AF228063).

Figure 4. Schematic of conserved protein motifs.

Conservation of LUJV amino acid motifs with respect to all other (green highlight), to OW (yellow highlight), or to NW (blue highlight) arenaviruses is indicated; grey highlight indicates features unique to LUJV. Polymerase motifs pre-A (L₁₁₄₂), A (N₁₂₀₉), B (M₁₃₁₃), C (L₁₃₄₅), D (Q₁₃₈₆), and E (C₁₃₉₈) are indicated for the L ORF; potential myristoylation site G₂, the RING motif H₃₄/C₇₆, and potential late domains YXXL an PSAP are indicated for the Z ORF; and myristoylation site G₂, posttranslational processing sites for signalase (S₅₉/S₆₀) and S1P cleavage (RKLM₂₂₁), CTL epitope (I₃₂), zinc finger motif P₄₁₅/G₄₄₀, as well as conserved cysteine residues and glycosylations sites (Y) are indicated for GPC. * late domain absent in NW viruses and DANV; † PSAP or PTAP in NW viruses, except in PIRV and TCRV (OW viruses: PPPY); # G in all viruses except LCMV ( = A); ‡ D in NW clade A only; § conserved with respect to OW, and NW clade A and C; HD, hydrophobic domain; TM, transmembrane anchor.