Hematoxylin and eosin stained sections through lung (A) and spleen (B) of infected M. schreibersii . (A) Thickened interalveolar septae (arrowhead) (bar = 500 m m) and infiltrates comprising lymphocytes and macrophages (higher magnification inset) (bar = 50 m m). (B) 

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... cause lethal hemorrhagic fever in humans and nonhuman primates. The family Filoviridae includes two genera: Marburgvirus , comprising various strains of the Lake Victoria marburgvirus (MARV); and Ebolavirus (EBOVs), comprising four species including Sudan ebolavirus (SEBOV), Zaire ebolavirus (ZEBOV), Ivory Coast ebolavirus (CIEBOV), and Reston ebolavirus (REBOV); and a tentative species Bundibugyo ebolavirus (BEBOV) [1]. MARV was discovered in 1967 in Marburg, Germany during an outbreak in laboratory staff exposed to tissues from monkeys imported from Uganda. ZEBOV was discovered in 1976 in Yambuku, Zaire during a 312-person outbreak associated with 90% mortality. With the exception of REBOV, that appears to be pathogenic in nonhuman primates but not in humans and is endemic in the Philippines, all known filoviruses are pathogenic in primates including humans and are endemic in Africa [2]. Bats are implicated as reservoirs and vectors for transmission of filoviruses in Africa [3]. ZEBOV sequences have been found in fruit bats ( Hypsignathus monstrosus, Epomops franqueti and Myonycteris torquata ) [4,5]. MARV sequences have been found in fruit ( Rousettus aegyptiacus ) and insectivorous ( Rhinolophus eloquens and Miniopterus inflatus ) bats [6,7]. Bats naturally or experimentally infected with ZEBOV or MARV are healthy and shed virus in their feces for up to 3 weeks [4,5,7]. In 2002, colonies of Schreiber’s bats ( Miniopterus schreibersii ), sustained massive die-offs in caves in France, Spain and Portugal [8]. M. schreibersii , family Vespertilionidae , comprises at least four geographically discrete lineages distributed in Oceania, southern Europe, southern Africa, and southeast Asia [9]. Here we report the discovery of a novel ebolavirus-like filovirus in bats from Europe. Bat carcasses from Cueva del Lloviu, Asturias, Spain (5 32 9 8.1 9 N and 43 u 30 9 5.6 9 W) were collected for anatomical, microbiological and toxicological analyses. Although no gross pathology was apparent, microscopy of internal organs revealed interstitial lung infiltrates comprised of lymphocytes and macrophages, and depletion of lymphocytes and lymphoid follicles in spleen ( Figure 1) . These findings were consistent with viral pneumonia; hence, nucleic acid from lung and spleen were analyzed by consensus polymerase chain reaction (PCR) for the presence of a broad range of viral agents including lyssa-, paramyxo-, henipa-, corona-, herpes- and filoviruses. Filovirus sequences were detected in extracts from lung, liver, rectal swabs or spleen of 5 animals. Pairwise distance analysis of the 186 nucleotide product showed highest similarity with ZEBOV (73.7%). A sensitive real time PCR assay established to quantitate viral burden confirmed the presence of filoviral sequences in the original 5 animals and from an additional 15 with similar pathology collected from the same cave ( Table 1 ). A liver sample with the highest viral load by PCR (4.0 6 10 6 genome copies/gr) was selected for high-throughput sequencing yielding 225,758 reads that represented 12.1 kilobases of viral sequence. Gaps between fragments and genomic termini were completed by specific PCR and rapid amplification of cDNA ends (RACE) to obtain a nearly complete genome. Reports of bat die-offs in additional caves prompted analysis of a second set of samples from caves in Cantabria, Spain, wherein many dead M. schreibersii were observed. Throat and rectal swabs, brain, lung and liver were collected from five dead M. schreibersii, and nine dead Myotis myotis . Whereas filovirus sequences were detected by real time PCR in all M. schreibersii samples, no filovirus sequences were found in the M. myotis ( Table 1 ). Real time PCR analysis of throat swabs and stool samples from 1,295 healthy bats representing 29 different bat species (including 45 healthy M. schreibersii from Lloviu cave collected after the die-offs) collected in several geographic locations in Spain revealed no evidence of filovirus infection ( Figure S1 ). Sequencing of regions of the L and NP genes of the original Lloviu Cave bat samples resulted in nearly identical sequences to the prototype sequence; a similar lack of variation was observed within each lineage of MARV in fruit bat reservoirs in the Kitaka cave, Uganda, although in that instance two clearly differentiated lineages were observed [7]. Consistent with the genomic organization characteristic of filoviruses, Lloviu virus (LLOV), named for the cave in which it first was found, has a 19 kb negative sense, single stranded RNA genome that contains seven open reading frames (ORF) (GenBank Accession number JF828358). However, LLOV differs from other filoviruses in transcriptional features. Analysis of conserved transcriptional initiation and termination sites suggests that the seven LLOV ORFs are encoded by six mRNA transcripts, one of which is dicistronic and contains both the VP24 and the L ORF ( Figure 2 ). Additionally, although the LLOV termination signal is identical to ebolaviruses, the LLOV initiation signal is unique (3 9 - CUUCUU(A/G)UAAUU-5 9 ). Several attempts by RACE to obtain complete genomic sequence were unsuccessful. By analogy to other filoviruses we assume that up to 700 nt may be missing at the 5 9 terminus of the genome. This assumption is based on the observation that all known negative-strand RNA viruses have complementary termini and that length of noncoding sequences at the termini of filoviruses do not exceed 700 nt. LLOV sequence was analyzed for similarities to EBOVs and MARV. In EBOVs a C-terminal basic amino acid motif in VP35 mediates type I interferon antagonism by binding to double- stranded RNA and inhibiting RIG-I signaling. This domain is conserved in LLOV VP35 ( Figure S2 ). In non-segmented, negative strand RNA viruses, matrix proteins are not only key structural components of the virions, but also play important roles in the maturation and cellular egress steps of the viral life cycles. Short amino-acid sequences, termed late-budding motifs or L domains, are crucial for these events. The matrix protein in EBOVs, encoded by VP40, has overlapping P(T/S)AP and PPXY late-budding motifs at the N-terminus [10,11] and YXXL late- budding motifs in the C-terminus. MARV VP40s contains only PPXY motifs. LLOV contains only a PPXY motif in the N- terminal domain of the VP40; hence, in this aspect, it is more similar to MARV than to EBOVs. The filovirus GP2 has an immunosuppressive motif [12,13] ( Figure S3 ); this motif is highly conserved in LLOV. EBOV VP24 interacts with the KPN a proteins that mediate PY-STAT1 nuclear accumulation [14]. Two domains of VP24 are required for inhibition of IFN- b -induced gene expression and PY-STAT1 nuclear accumulation (region 36– 45 and 142–146) [15]. LLOV VP24 ORF has significant homology to EBOV VP24s; however, interaction domains are not well conserved ( Figure S4 , shaded areas). Phylogenetic analysis of conserved domain III of the RNA- dependent RNA polymerase demonstrates that LLOV belongs to the Filoviridae and may represent a complex of viruses related to all EBOVs ( Figure 3A ). Phylogenetic analysis of complete genome ...