Phylogenetic and genomic analysis of EVEs across multiple host species revealed novel information about the origin and evolution of diverse virus groups. Furthermore, several of the elements identified here encode intact open reading frames or are expressed as mRNA. For one element in the primate lineage, we provide statistically robust evidence for exaptation. Our findings establish that genetic material derived from all known viral genome types and replication strategies can enter the animal germ line, greatly broadening the scope of paleovirological studies and indicating a more significant evolutionary role for gene flow from virus to animal genomes than has previously been recognized.Integration into the nuclear genome of germ line cells can lead to vertical inheritance of retroviral genes as host alleles. For other viruses, germ line integration has only rarely been documented. Nonetheless, we identified endogenous viral elements (EVEs) derived from ten non-retroviral families by systematic  The presence of retrovirus sequences in animal genomes has been recognized since the 1970s, but is readily explained by the fact that these viruses integrate into chromosomal DNA as part of their normal replication cycle. Unexpectedly, however, we identified a large and diverse population of sequences in animal genomes that are derived from non-retroviral viruses. Analysis of these sequences—which represent all known virus genome types and replication strategies—reveals new information about the evolutionary history of viruses, in many cases providing the first and only direct evidence for their ancient origins. Additionally, we provide evidence that the functionality of one of these sequences has been maintained in the host genome over many millions of years, raising the possibility that captured viral sequences may have played a larger than expected role in host evolution. However, they can also increase in frequency, and some eventually reach fixation Viral infection of germ line cells  can lead to viral genes or genomes becoming integrated into chromosomes and inherited as host alleles In animal genomes, the majority of EVEs are derived from reverse transcribing RNA (rtRNA) viruses (i.e. retroviruses) exapted retroviral genes  in mammalian reproduction EVEs reveal complex evolutionary relationships between viruses and their hosts. For example, endogenous retroviruses have shaped vertebrate genome evolution, not only by acting as genetic parasites EVEs also constitute an invaluable resource for reconstructing the long-term history of virus and host evolution in silico approach to screen the genomes of mammals, birds and insect vector species for endogenous sequences derived from non-retroviral mammalian viruses. We identify sequences derived from a very broad range of viruses, revealing an extensive history of non-retroviral genome invasion ranging back to at least the late Mesozoic Era (∼93 million years ago). We demonstrate that these sequences can be highly informative; (i) revealing novel virus diversity; (ii) providing a timescale for virus evolution; (iii) indicating the likely host range of virus groups, and; (iv) identifying rare instances of horizontal transmission. Furthermore, using a novel approach, we show that at least some of the EVE sequences identified here are likely to have been exapted during their evolution. The implications of these findings are discussed.Despite the large quantity of published genome sequence data, the diversity of non-retroviral viruses in animal genomes has not been systematically explored. In this report, we use an in silico screening of genomes for endogenous non-retroviral insertions was developed. We selected all non-retroviral virus genera that infect mammals, and constructed a library of representative peptide sequences ) ) . The tBLes (Kb)) . Sequences (Kb)) , S6, S7.−9) to RNA viruses in the genomes of mammals and insect vectors (Reoviridae) and positive sense RNA (RNA+ve) viruses (Flaviviridae), as well as both segmented  and non-segmented  families of negative sense RNA (RNA-ve) viruses. Consistent with an integration process involving viral mRNA (rather than genomic RNA), all EVEs derived from RNA viruses had genetic structures that spanned a single viral transcript (or fragments derived from single transcripts). EVEs derived from different genes never occurred as contiguous sequences, and consequently we could not determine whether EVEs derived from distinct genes of a given virus family originated from the same or distinct virus lineages/infections.We identified numerous, highly significant matches  and 3′ poly-A tails, consistent with LINE-mediated retrotransposition of viral mRNAs Mononegavirales  were identified. Matches to genes encoding the relatively conserved nucleoprotein (NP) and L-polymerase proteins predominated, but matches to more rapidly evolving glycoproteins were also identified (Numerous EVE sequences disclosing similarity to proteins derived from non-segmented RNA-ve virus families in the order entified . A subseentified  exhibiteEchinops telfairi)). Bornavirus nucleoprotein trees revealed an expansion of EBLNs in the New World branch of strepsirrhine primates, represented by the common marmoset   elements jacchus) .Myotis lucifugus), Tammar wallaby (Macropus eugenii), and gray short-tailed opossum (Monodelphis domestica) genomes. The majority of these matches comprised fragments of genes, although two full-length NP gene EVEs (displaying poly-A tails and TSDs) were identified in bats (Dipodymys ordii), guinea pig (Cavia porcellus) and common shrew (Sorex araneus) genomes. Additionally, fragments of the L-polymerase and VP35 gene were identified in the genomes of the opossum and Philippine tarsier (Tarsius syrichta) respectively. In phylogenies, EVEs derived from filovirus NP genes grouped into two well-supported clades , and in the genomes of both Aedes and Culex mosquitoes. Among these were insertions that encoded intact NP and GP ORFs (Drosophila sigma virus (Aedes and Culex was high (96%). A robust clade (100% bootstrap support) placed four Ixodes EVEs into a single group, suggesting they are likely derived from the same exogenous virus lineage, but their placement relative to other Rhabdoviruses was ambiguous, as they formed a clade with a number of distinct Rhabdoviruses with minimal bootstrap support (8%). Phylogenies constructed using L-polymerase sequences weakly grouped Ixodes and Aedes insertions with Lyssaviruses and Moussa virus, but not Drosophila sigma virus. Weak support for basal relationships was obtained with both trees, making it difficult to confidently place thenovel EVEs with respect to the known rhabdovirus diversity.EVEs related to rhabdoviruses were identified in the black-legged tick genome  Orthomyxoviridae (Bunyaviridae (Phlebovirus and Nairovirus genera) were identified in the I. scapularis genome. Nairovirus-derived EVEs were distantly related to Hazara virus  to segment 5 of the Liaoning virus genome  that was recently isolated from Aedes dorsalis mosquitoes A. aegyptii had a large inframe deletion, but encoded an otherwise intact ORF. This is the first EVE derived from a dsRNA virus to be described. As with other RNA virus EVEs in insect genomes, the mechanism of genomic integration was unclear. The intact ORFs and high level of identity to a circulating virus raise the possibility this EVE formed recently and is not fixed in the host population.In the s genome . LiaoninAedes mosquito contains several sequences exhibiting similarity to the viruses of the RNA+ve family Flaviviridae. Endogenous flaviviruses have previously been reported in the genomes of A. aegyptii and A. albopictus mosquitoes A. albopictus, and a range of smaller fragments at the 3′ end of the flaviviral genome, mostly of the NS5 gene, have been described in both albopictus and aegyptii species. We have identified fragments that together span almost the entire flavivirus genome in A. aegyptii , includParvoviridae and Circoviridae - the first ssDNA virus EVEs to be described in mammals - and one family of reverse transcribing DNA (rtDNA) viruses (Hepadnaviridae) - the first rtDNA EVEs to be described. A single match to a double stranded DNA (dsDNA) virus family (Adenoviridae) was identified in the kangaroo rat genome, but this sequence was unambiguously viral across its entire length (∼17 Kb), encoding thirteen completely intact viral ORFs , and the zebrafinch (Taeniopygia guttata). In the zebrafinch genome, a total of 7 loci were identified on 7 distinct chromosomes  genome matched the Amdovirus genus (EVEs derived from viruses of the family  genomes . In totaus genus . Phylogeus genus .Loxodonta africana) encoded an intact NS1 gene (Trichosurus vulpecula).The majority of parvovirus EVEs were not intact, and are unlikely to express RNA or protein. However, a dependovirus EVE in the genome of the African elephant (NS1 gene . AdditioAdeno-associated virus 2 (AAV-2) integrates at a specific site in human chromosome 19 Circoviridae were identified in the genomes of the gray short-tailed opossum, and three species of the mammalian order Carnivora; domestic cat (Felis catus), dog (Canis familiaris) and panda (Ailuropoda melanoleuca) (EVEs disclosing significant similarity to the Rep gene of ssDNA family noleuca) . These snoleuca) . One cirParvo, Circo and Bornaviridae of 30, 68 and 93 million years respectively, demonstrating the ancient origins of these families  with respect to their known range as exogenous viruses (in vivo models M. lucifugus) and Asian primates (tarsier), but also in insectivores, rodents, and in both South American and Australian mammals  genome grouped robustly with avian dependoviruses  in NS1 trees (Highly discordant host ranges among closely related EVEs (or EVEs and exogenous viruses) can provide information about transmission events. In this regard, we note that a dependovirus EVE in the bottlenose dolphin  Richer sampling of animal genomes is likely to reveal an even greater diversity of EVEs. While EVEs that are very ancient (i.e. that inserted prior to the divergence of major host lineages) can be identified by selectively screening a small number of host species, identification of more recent insertions will often require richer sampling within orders and genera. Sampling of mammalian species for whole genome sequencing has generally been across, rather than within orders (primates are an exception). Consequently the majority of mammal species sampled in this study diverged more than 50 million years ago . Any mamin silico screening strategy employed here likely underestimates the actual diversity of EVEs for several reasons. Firstly, only low-coverage, incomplete genome data were available for most species. Furthermore, EVEs within the data we screened could have been overlooked because (i) screening was based on similarity searches, and is thus dependent on current (limited) knowledge of viral diversity, and (ii) more ancient EVEs may not be identified due to the divergence in both host and virus lineages subsequent to insertion .The Parvoviridae, Mononegavirales). This likely reflects a predisposition for germline integration among viruses with particular patterns of replication and infection. Notably, viruses that establish persistent infections and/or replicate within the nucleus are particularly well represented among the EVEs identified in this study. Nevertheless, these characteristics do not appear to be prerequisites for germ line integration  viruses appear to be better represented in the genomic fossil record than others  master species list (−9) to host proteins; or (iii) non-viral and highly repetitive (>50 copies per host genome). Insertions were considered endogenous when >100 bp of genomic flanking sequence could be identified either side of a viral match. Insertions for which >100 bp of unambiguous (i.e. >80% nucleotide identity) flanking sequence was identified in host sister taxa were considered orthologous insertions. PERL scripts were used to automate BLAST searches and sequence extraction. Putative EVE peptide sequences, and alignments of EVEs and exogenous retroviruses, are available online .Chromosome assemblies and whole genome shotgun assemblies of 44 species  were scries list ). Host gParvoviridae: dependovirus NS1 gene , Parvoviridae: parvovirus NS1 gene, , Circoviridae: Rep gene , Hepadnaviridae: polymerase gene , Orthomyxoviridae: GP gene , Reoviridae: VP5 gene , Bunyaviridae: phlebovirus NP gene , Bunyaviridae: nairovirus NP gene , Flaviviridae: mostly NS3 gene , Filoviridae: NP gene , Filoviridae: L gene , Bornaviridae: NP gene , Bornaviridae: L gene , Rhabdoviridae: NP gene , Rhabdoviridae: L gene .Putative EVE sequences inferred using BlastAlign were aligned with closely related viruses using MUSCLE and manually edited et alA Monte Carlo simulation procedure was employed to determine the probability that the bornavirus-derived element EBLN-1 has retained coding capacity over 54.1 million years under neutral evolution (i.e. not under purifying selection). A consensus EBLN-1 sequence was inferred, and the effects of neutral evolution were simulated using seq-gen Parvoviridae; AAV2 (NC_001401); Minute virus of mice (NC_001510.1); AMDV (NC_001662); Goose parvovirus (EU583390.1); Muscovy duck parvovirus (X75093.1); Porcine hokovirus (EU200671.1); Snake parvovirus (AY349010.1); Avian AAV ; AAV1 (AF063497.1); AAV4 (U89790); AAV2 (AY695375.1); Bovine AAV (AY388617.1); Caprine AAV (DQ335246.2); Bocavirus (M14363.1); Erythrovirus (AB126265.1); Aleutian mink disease virus (M20036.1); Porcine parvovirus (EU790642.1); Feline panleukopenia virus (EF988660.1); Canine parvovirus (EU310373.2); Rat parvovirus (AF036710.1); Hamster parvovirus (U34255.1); Minute virus of mice (DQ196317.1); Kilham rat virus (U79033.1); Circoviridae; Porcine circovirus 1 (NC_006266); Porcine circovirus 2 (GU325757); Cyclovirus PK5006 (GQ404856.1); Cyclovirus NG14 (GQ404855.1); Human stool-associated circular virus NG13 (GQ404856.1); Beak and feather disease virus (AY450436.1); Columbid circovirus (AF252610.1); Hepadnaviridae; duck HBV (NC_001344); Stork HBV (AJ251937.1|); Heron HBV (NC_001486); Ross' Goose HBV (AY494849.1); Crane HBV (AJ441113.1); Sheldgoose HBV (AY494852.1); Snow goose HBV (AF111000.1); Woodchuck HBV (AF410861.1); Flaviviridae; Kamiti river virus (NC_005064); Aedes flavivirus (NC_012932); Quang binh virus (NC_012671); Culex flavivirus (NC_008604); Nakiwogo virus (GQ165809). Reoviridae; Liaoning virus (NC_007736 - NC_007747); Kadipiro virus ; Banna virus . Bunyaviridae; Crimean-Congo hemorrhagic fever virus ; Uukuniemi virus ; Uukuniemi virus (M33551.1); Catch-me-cave virus (EU274384.1); Sandfly fever Naples virus (EF201832.1); Massilia virus (EU725773.1); Punta Toro virus (EF201834.1); Buenaventura virus (EF201839.1); Rift Valley fever virus (DQ380156.1); Phlebovirus sp. (EF201818.1); Icoaraci virus (EF076014.1). Orthomyxoviridae; Quaranfil virus (FJ861694.1); Johnston Atoll virus (FJ861696.1); Thogoto virus (M77280.1); Dhori virus (M34002.1). Bornaviridae; Borna disease virus (NC_001607); Avian BDV (FJ169441). Filoviridae; Reston ebola virus (NC_002549); Zaire ebola virus (NC_002549); Lake Victoria marburgvirus (NC_001608). Rhabdoviridae; vesicular stomatitis virus (NC_001560); Wongabel virus (NC_011639); Kotonkon virus (DQ457099); Adelaide river virus (U10363.1); Obodhiang virus (DQ457098.1); Bovine ephemeral fever virus (AF234533.1); Rochambeau virus (DQ457104.1); Mount elgon bat virus (DQ457103.1); Oita rhabdovirus (AB116386); Kern canyon virus (DQ457101.1); Sandjimba virus (DQ457102.1); Kolongo virus (DQ457100.1); Tupaia rhabdovirus (AY840978.1); Spring viremia of carp (DQ491000.1); Pike fry rhabdovirus (FJ872827.1); Cocal virus (EU373657.1); Vesicular stomatitis Indiana virus (AF473865.1); Isfahan virus (AJ810084.2); Chandipura virus (AY614728.1); Ngaingan virus (FJ715959.1); Wongabel virus (EF612701.1); Flanders virus (AF523194.1). Nyaviridae; Midway virus (NC_012702); Nyamanini virus (NC_012703).Figure S1Aedes aegyptii genome and Liaoning virus segment 5. Genomic regions, as determined by alignment to a repetitive element (RE) in the A. aegyptii genome, are indicated in blue, on coding viral regions are shown in red, and regions encoding viral proteins are shown in green.Sequence alignment of an EVE identified in the (0.40 MB PDF)Click here for additional data file.Figure S2Dipodymys ordii). The name of the corresponding protein in the most closely related virus  is indicated above each open reading frame (ORF). Arrows beneath ORFs indicate frames encoded in reverse direction relative to contig. Abbreviations: kd = kiloDalton; pol = DNA polymerase; T = terminal protein; P = penton base; Mco = minor core; Mca = minor capsid; DB = DNA binding; Ma = Major coat; H = hexon-associated; S = shaft.Genetic structure of an adenovirus related sequence identified in whole-genome shotgun sequence data for Ord's kangaroo rat ((0.27 MB PDF)Click here for additional data file.Figure S3Myotis lucifugus) genome. The element is a composite of two genomic contigs, which were assembled by identifying the empty pre-integration site in the closest relative (Pteropus vampyrus). The inset box shows an alignment the inverted repeats in the 5′ and 3′ untranslated regions. Abbreviations: IR = inverted repeat. NS1 = Non-structural protein 1; VP2 = Viral protein 2; UTR = untranslated region.Genetic structure of a complete dependovirus genome identified in the little brown bat ((0.32 MB PDF)Click here for additional data file.Table S1Genome sequences screened for endogenous viral elements.(0.09 MB DOC)Click here for additional data file.Table S2in silico screening of host genomes.Viral reference sequences used for (0.14 MB DOC)Click here for additional data file.Table S3Endogenous viral elements related to negative sense RNA viruses.(0.40 MB DOC)Click here for additional data file.Table S4Endogenous viral elements related to doubled-stranded RNA viruses.(0.03 MB DOC)Click here for additional data file.Table S5Endogenous viral elements related to positive sense RNA viruses.(0.09 MB DOC)Click here for additional data file.Table S6Endogenous viral elements related to single stranded DNA viruses.(0.21 MB DOC)Click here for additional data file.Table S7Endogenous viral elements related to reverse transcribing DNA viruses.(0.04 MB DOC)Click here for additional data file.


Aedes albopictus, trapped around the nearby French military camp. The Chikungunya virus was isolated from mosquitoes and found to be identical to the A226V circulating human strain. This is the first field study showing the role of the recently arrived species Aedes albopictus in Chikungunya virus transmission in Central Africa, and it demonstrates this species' role in modifying the epidemiological presentation of Chikungunya in Gabon.The primary vector at the origin of the 2007 outbreak in Libreville, Gabon is identified as  Alphavirus member of the Togaviridae, is now a world-wide public health problem Ae. albopictus was identified as the major vector in La Reunion Island and Europe Ae. albopictusFirst isolated in Tanzania in 1952, Chikungunya virus (CHIKV), an Togaviridae, Flaviviridae and BunyaviridaeAedes sp. including Aedes africanus, Aedes furcifer, Aedes luteocephalus, Aedes neoafricanus, Aedes tayloriAedes albopictus has succeeded in colonizing some parts of sub-Saharan Africa. In central Africa, this spreading vector was first collected in 2000 in Cameroon, in 2003 in Equatorial Guinea, and in 2006 in Gabon Aedes sp. such as Aedes aegypti or the recently discovered Aedes albopictus as a primary vector.In Africa, while recent serological surveys suggest a high prevalence of Aedes albopictus was first detected in December 2006. The majority of patients were presumptively treated for malaria with no symptom improvement. When malaria diagnosis tests  were performed, the results were negative and CHIKV was identified from patient blood samples as the main causative agent Aedes albopictus adult abundance and oviposition rate are closely correlated with rainfall In April 2007, an outbreak of Dengue-like symptoms occurred in Libreville where Aedes albopictus and Aedes aegypti are potential vectors of CHIK and DEN viruses. To assess the respective roles of Aedes albopictus and Aedes aegypti in Dengue and chikungunya transmission in Libreville, we proceeded in two steps. An experimental study was conducted in the laboratory, using experimental infections to determine the ability of Ae. albopictus and Ae. aegypti collected in Libreville to transmit CHIK and Dengue virus Ae. albopictus showed a higher susceptibility to CHIK virus than Ae. aegypti. Thus, Ae. albopictus appears to be a better vector for CHIK virus than Ae. aegypti. Moreover, as demonstrated by the rates of disseminated infection obtained, Gabonese Ae. albopictus was less susceptible to DEN virus, leading us to consider Ae. albopictus as a secondary dengue vector. During the same period, a field study was conducted to research DENV- and CHIKV-infected mosquitoes. The objective of the present study is to identify the primary vector at the origin of the Libreville outbreak.In Libreville, Aedes sp. caught inside and outside the camp were compared using Student's t-test. Each day's collections of Aedes mosquitoes were sorted by trap, species and sex. Mosquito pools were mixed with sterile PBS, crushed and 0.22 µm filtered . For samples of fewer than 5 mosquitoes, a PBS volume of 200 µl was used for crushing; for larger samples, the volume was increased to prevent the mixture becoming too viscous. Genomic CHIKV detection was performed for each pool using TaqMan RT-PCR Bg-sentinel odor traps  were used in the French military camp of Libreville (lat: 0.44 long: 9.43) and its neighboring areas . Traps wCulex quinquefasciatus , Mansonia uniformis , Anopheles gambiae , Anopheles sp. , Aedes sp. , 1046 Aedes albopictus  and 80 Aedes aegypti . The average number of Ae. albopictus specimens per trap caught inside and outside the camp were 1.7 and 16.3, respectively (p<0.0001). There was no statistical difference between the average number of Ae. Aegypti specimens caught inside and outside the camp. Pool sizes and compositions are given in Aedes aegypti nor Aedes albopictus pools were positive for Dengue virus. The two Aedes albopictus female pools  were positive for CHIKV according to genome detection by TaqMan RT-PCR, and Chikungunya virus was also isolated when their filtered supernatants were inoculated onto Vero E6 and C6/36 cell lines. The female Aedes albopictus minimal infection rate  was approximately 2.5‰. The CHIKV genomes were partially sequenced (GenBank accession number EU403052). When compared to the human isolates from blood samples of Gabonese patients Aedes albopictus isolated CHIKV displayed 98.7% to 99.1% identity at the nucleotide level and 99.4% to 99.6% identity at the amino-acid level. Both human and mosquito isolates displayed the A226V mutation within the E1 gene During the two weeks studied in July 2007, a total of 10,605 mosquitoes were caught: Ae. albopictusAedes albopictus strain. Interestingly, an analysis of full-length viral sequences reveals three independent instances of Ae. albopictus exposure to the virus in India, Cameroon and Gabon, each followed by the acquisition of a single adaptive mutation providing a selective advantage for transmission by this mosquito Sequence analysis of the virus genome revealed that the recent CHIKV outbreaks were caused by a new variant characterized by a mutation in the E1 envelope glycoprotein gene (A226V) Aedes mosquitoes is based on the destruction or elimination of unwanted natural and artificial water containers. The efficacy of this strategy is highlighted by the lower average number of Ae. albopictus caught in traps inside the camp. This study confirms that reducing the number of such containers in and around homes significantly decreases the size of Ae. albopictus populations.Inside the camp, the vector control program against Aedes aegypti captured did not allow us to determine if this species had participated as a second vector for CHIKV transmission during the outbreak. In Libreville, Ae. aegypti and Ae. albopictus are often sampled in the same container Ae. albopictus exploits a wider range of breeding sites than Ae. aegypti, which is classically more dependent on artificial breeding sites than Ae. albopictus. During the dry season, the reduction of artificial breeding sites has a stronger impact on Ae. aegypti population size. Our field study is not able to clarify the role of Ae. aegypti in CHIKV transmission during the outbreak nor to identify Ae. aegypti as the DEN vector in Libreville. Nevertheless, the results of experimental infection suggest that Ae. aegypti was probably a better DEN vector in Libreville Ae. albopictus was a more efficient experimental vector of CHIK virus than Ae. aegypti. formosus, and is assumed to be a better CHIK vector in the field, not playing a significant role in DEN transmission in Gabon.The low number of Aedes albopictus during the middle of the dry season and the high experimental infection rate highlight the role of this mosquito as the primary vector during the 2007 outbreak in Gabon. This was the first field study showing the role of recently arrived Aedes albopictus in the transmission of Chikungunya virus in Central Africa. Aedes albopictus has currently been collected in four of the Gabon's nine provinces, indicating an ongoing colonization of the country similar to the colonization of Cameroon Aedes albopictus in the modification of Chikungunya epidemiological presentation in Gabon from rural and endemic to urban and endemo-epidemic. This trend was observed as early as 2000 in outbreaks in the Democratic Republic of the Congo Ae. albopictus continues to spread, displacing Ae. aegypti in African countries like Cameroon Ae. albopictus should not be an efficient DEN vector The high level of identity between the human and mosquito virus isolates from Libreville indicates a strong relationship between these CHIKV isolates, while the finding of CHIKV infected