Stranded RNA Genome Research Articles

Emaravirus-specific degenerate PCR primers allowed the identification of partial RNA-dependent RNA polymerase sequences of Maize red stripe virus and Pigeonpea sterility mosaic virus

Emaravirus is a recently established viral genus that includes two approved virus species: European mountain ash ringspot-associated virus (EMARaV) and Fig mosaic virus (FMV). Other described but unclassified viruses appear to share biological characteristics similar to emaraviruses, including segmented, negative-single stranded RNA genomes with enveloped virions approximately 80-200nm in diameter. Sequence analysis of emaravirus genomes revealed the presence of conserved amino acid sequences in the RNA-dependent RNA polymerase gene (RdRp) denoted as pre-motif A, motifs A and C. Degenerate oligonucleotide primers were developed to these conserved sequences and were shown to amplify in reverse transcription-polymerase chain reaction assay (RT-PCR) DNA fragments of 276bp and 360bp in size. These primers efficiently detected emaraviruses with known sequences available in the database (FMV and EMARaV); they also detected viruses with limited sequence information such as Pigeonpea sterility mosaic virus (PPSMV) and Maize red stripe virus (MRSV). The degenerate primers designed on pre-motif A and motif A sequences successfully amplified the four species used as positive controls (276bp), whereas those of motifs A and C failed to detect only MRSV. The amino acid sequences obtained from PPSMV and MRSV shared the highest identity with those of two other tentative species of the Emaravirus genus, Rose rosette virus (RRV) (69%) and Redbud yellow ringspot virus (RYRV) (60%), respectively. The phylogenetic tree constructed with 92 amino acid-long portions of polypeptide putatively encoded by RNA1 of definitive and tentative emaravirus species clustered PPSMV and MRSV in two separate clades close to RRV and Raspberry leaf blotch virus (RLBV), respectively. The newly developed degenerate primers have proved their efficacy in amplifying new emaravirus-specific sequences; accordingly, they could be useful in identifying new emaravirus-like species in nature.

Structure-Activity Relationships in the Development of Allosteric Hepatitis C Virus RNA-Dependent RNA Polymerase Inhibitors: Ten Years of Research

Hepatitis C is a viral liver infection considered as the major cause of cirrhosis and hepatocellular carcinoma (HCC). Hepatitis C virus (HCV) possesses a single positive strand RNA genome encoding a polyprotein composed of approximatively 3000 amino acids. The polyprotein is cleaved at multiple sites by cellular and viral proteases to liberate structural and nonstructural (NS) proteins. NS5B, the RNA-dependent RNA polymerase (RdRp), which catalyzes the HCV RNA replication has emerged as an attractive target for the development of specifically targeted antiviral therapy for HCV (DAA, for direct-acting antivirals). In the last 10 years, a growing number of non-nucleoside compounds have been reported as RdRp inhibitors and few are undergoing clinical trials. Over the past 5 years, several reviews were published all describing potentially active molecules. To the best of our knowledge, only one review covers the structure-activity relationships.(1) In this review, we will discuss the reported non-nucleoside molecules acting as RdRp inhibitors according to their chemical class especially focusing on structure-activity relationship aspects among each class of compounds. Thereafter, we will attempt to address the global structural requirements needed for the design of specific inhibitors of RdRp.

Selection and evaluation of single domain antibodies toward MS2 phage and coat protein

MS2 phage (MS2 Ø) is a coli phage, non-pathogenic to eukaryotic cells, which has been used as a simulant for viral biothreats, such as those causing smallpox and hemorrhagic fever. MS2 Ø consists of an icosahedral capsid, 28nm in diameter, and a single stranded RNA genome; the viral capsid is composed of 180 copies of coat protein (CP). In this study, we isolated anti-MS2 Ø single domain antibodies (sdAbs) for the sensitive detection of the MS2 Ø. To achieve this, a first immune sdAb library was prepared from llamas immunized with purified coat protein and a second from animals immunized with MS2 Ø. By panning the two libraries against CP, MS2 Ø, or alternating between the two targets, anti-MS2 Ø and anti-CP sdAbs were selected, sequenced, and characterized for their binding affinity. Both direct binding assays and capture sandwich assays were performed on the MAGPIX platform. One of the best anti-MS2 Ø sdAb, Lib2CP12H, could detect MS2 Ø concentrations as low as 1.45ng/mL (∼5.0E+6pfu/mL), providing equivalent detection to conventional antibodies. This sdAb is thermally stable with a melting temperature around 60°C and recovered 80% of its secondary structure after heat denaturation.

La protéine L des Mononegavirales.

The order Mononegavirales comprises viruses representing a large variety of human, animal and plant pathogens. These viruses have a non-segmented negative stranded RNA genome encoding their own RNA dependent RNA polymerase, the large protein L used to transcribe and replicate their genome. Due to the absence of methods to produce purified recombinant L proteins, these later were poorly characterized. Recently, the vesicular stomatitis virus L protein has been purified and used for structural and functional analysis. Electron microscopy images of L provided the first structural insight into the molecular architecture of L, and its conserved regions were mapped. Moreover, a new in vitro assay was developed and the mechanism by which L initiates RNA synthesis has been characterized, as well as the effects of its co-factor, the phosphoprotein P, on its own activity. This system constitutes a powerful tool to screen inhibitors directed specifically against the polymerase activity of L. These data have led to significant progress into the understanding of how the replicative machinery of these viruses function.

Current Status of Bluetongue Disease, Its Vector and Pathogenesis in India

Bluetongue (BT) is a Culicoides (Diptera: Ceratopogonidae) borne, infectious, noncontagious, economically important viral disease of sheep and other domestic and wild ruminants. Humans are not affected although some species of Culicoides feed on human blood also. The disease has the potential to spread rapidly, which can cause serious socio-economic consequences in view of mandatory trade barrier on the movement of animals, their germplasm, embryo and other animal products from BT endemic to BT free countries. The first outbreak of BT in sheep and goats in the country was recorded in 1964 in Maharashtra State. Since then, several outbreaks of BT have been reported in sheep in almost all states of the India. A serological survey has indicated the presence of bluetongue virus (BTV) antibodies in the Indian cattle, buffalos, goats, camels and some wild ruminants. However, clinical BT has not been observed in cattle or buffalo to date, whereas in goats, there are sporadic reports of occurrence of clinical BT either alone or as co-infection with peste des pestis ruminants. Although BTV has been isolated from Culicoides midges, the particular species responsible for transmission has not yet been identified. BTV is the prototypic member of the genus Orvivirus, family Reoviridae. BTV a non enveloped icosahedral virus consists of a ten segmented, double stranded RNA genome encapsidated in a double layered protein coat. Immunization programme is in progress in India and multivalent inactivated adjuvant vaccine containing BTV serotypes 1, 2, 10, 16 and 23 has been developed. Discovery of VLPs and CLPs has opened a new era of vaccination. The vector of BT is the Dipteran spp. Culicoides. About 60 spp. of Culicoides are reported from India. The early ecological study indicates that Culicoidesmonocoli Maheshwari may be the potential vector. The studies on BT in India are in primary stages, which may open several of vital fields for future investigation.

Stimulation of Hepatitis C Virus RNA translation by microRNA-122 occurs under different conditions in vivo and in vitro

Translation of the Hepatitis C Virus (HCV) positive strand RNA genome is directed by an internal ribosome entry site (IRES) in the viral RNA's 5′-untranslated region (5′-UTR). HCV propagates preferentially in the liver, and HCV translation is stimulated by the liver-specific microRNA-122 (miR-122) acting on two target sites in the 5′-UTR. This stimulation is effective in living cells containing miR-122 and also in the rabbit reticulocyte lysate in vitro-translation system after addition of miR-122. Another RNA sequence located in the Core protein coding sequence can base-pair in a long-range RNA–RNA interaction to the HCV 5′-UTR, overlapping with the miR-122 target sites and the short spacer between them, and thereby inhibits HCV translation. Here we show genetic evidence that in reticulocyte lysate single-stranded miR-122 interferes with this inhibitory long-range RNA–RNA interaction and thereby contributes to enhanced HCV translation, involving not only the 5′-seed sequence of miR-122 but also sequences at its 3′-end. Also RNA oligonucleotides shorter than a typical microRNA stimulate HCV translation, confirming that in the reticulocyte lysate the stimulation of HCV translation functions by displacement of the inhibitory long-range interaction by miR-122. In contrast, in transfected HuH-7 hepatoma cells and in HeLa cells this interference of miR-122 with the inhibitory long-range RNA–RNA interaction plays not a major role, but only duplex miR-122 RNAs of the correct length stimulate HCV translation. These results suggest that: (1) the processing of the microRNA precursors and (2) the events occurring at the HCV RNA differ between cells and reticulocyte lysate.

Molecular dynamics studies of the nucleoprotein of influenza A virus: role of the protein flexibility in RNA binding.

The influenza viruses contain a segmented, negative stranded RNA genome. Each RNA segment is covered by multiple copies of the nucleoprotein (NP). X-ray structures have shown that NP contains well-structured domains juxtaposed with regions of missing electron densities corresponding to loops. In this study, we tested if these flexible loops gated or promoted RNA binding and RNA-induced oligomerization of NP. We first performed molecular dynamics simulations of wt NP monomer and trimer in comparison with the R361A protein mutated in the RNA binding groove, using the H1N1 NP as the initial structure. Calculation of the root-mean-square fluctuations highlighted the presence of two flexible loops in NP trimer: loop 1 (73–90), loop 2 (200–214). In NP, loops 1 and 2 formed a 10–15 Å-wide pinch giving access to the RNA binding groove. Loop 1 was stabilized by interactions with K113 of the adjacent β-sheet 1 (91–112) that interacted with the RNA grove (linker 360–373) via multiple hydrophobic contacts. In R361A, a salt bridge formed between E80 of loop 1 and R208 of loop 2 driven by hydrophobic contacts between L79 and W207, due to a decreased flexibility of loop 2 and loop 1 unfolding. Thus, RNA could not access its binding groove in R361A; accordingly, R361A had a much lower affinity for RNA than NP. Disruption of the E80-R208 interaction in the triple mutant R361A-E80A-E81A increased its RNA binding affinity and restored its oligomerization back to wt levels in contrast with impaired levels of R361A. Our data suggest that the flexibility of loops 1 and 2 is required for RNA sampling and binding which likely involve conformational change(s) of the nucleoprotein.

Oligomerization paths of the nucleoprotein of influenza A virus

The influenza viruses contain a segmented, negative strand RNA genome. Each RNA segment is covered by multiple copies of the nucleoprotein (NP) and is associated with the polymerase complex into ribonucleoprotein (RNP) particles. Despite its importance in the virus life cycle, the interactions between the NP and the genome are not well understood. Here, we studied the assembly process of NP-RNA oligomers and analyzed how the oligomeric/monomeric status of RNA-free NP affects RNA binding and oligomerization. Recombinant wild-type NP purified in low salt concentrations and a derived mutant engineered for oligomerization deficiency (R416A) were mainly monomeric in RNA-free solutions as shown by biochemical and electron microscopy techniques. NP monomer formed with RNA a fast 1/1 complex characterized by surface plasmon resonance. In a subsequent and slow process that depended on the RNA length, oligomerization of NP was mediated by RNA binding. In contrast, preparations of wild-type NP purified in high salt concentrations as well as mutant Y148A engineered for deficiency in nucleic acid binding were partly or totally oligomeric in RNA-free solutions. These trimer/tetramer NP oligomers bind directly as oligomers to RNA with a higher affinity than that of the monomers. Both oligomerization routes we characterized could be exploited by cellular or viral factors to modulate or control viral RNA encapsidation by NP.

Hepatitis C virus entry into the hepatocyte.

Hepatitis C virus (HCV) is a small enveloped virus with a positive stranded RNA genome belonging to the Flaviviridae family. The virion has the unique ability of forming a complex with lipoproteins, which is known as the lipoviroparticle. Lipoprotein components as well as the envelope proteins, E1 and E2, play a key role in virus entry into the hepatocyte. HCV entry is a complex multistep process involving sequential interactions with several cell surface proteins. The virus relies on glycosaminoglycans and possibly the low-density lipoprotein receptors to attach to cells. Furthermore, four specific entry factors are involved in the following steps which lead to virus internalization and fusion in early endosomes. These molecules are the scavenger receptor SRB1, tetraspanin CD81 and two tight junction proteins, Claudin-1 and Occludin. Although they are essential to HCV entry, the precise role of these molecules is not completely understood. Finally, hepatocytes are highly polarized cells and which likely affects the entry process. Our current knowledge on HCV entry is summarized in this review.

Genome sequence of SeIV-1, a novel virus from the Iflaviridae family infective to Spodoptera exigua

Analysis of the transcriptome of Spodoptera exigua larvae revealed the presence of several ESTs with homology to virus of the order Picornavirales and with the highest similarity to Infectious flacherie virus (Iflaviridae) that infects Bombyx mori larvae. Iflaviridae is a recently defined family of insect-infecting viruses that consist of positive single strand RNA genomes translated into a single polyprotein of around 3000 amino acids long. Using the sequence information derived from the obtained ESTs, we have completed the genomic sequence of this virus. The novel S. exigua iflavirus (SeIV-1) has a genome of 10.3kb and codes for a 3222 aa polyprotein. Expression analysis has revealed the presence of the virus in all tissues tested and insect stages, being more abundant in the midgut of the larvae. High infectivity of this virus against S. exigua has been demonstrated after observing the presence of this virus in different colonies that were reared in the same chamber with the virus-infected colony, despite no evidence of pathological effects. Further study of viral covert infections of SeIV-1 could lead to a better understanding of its pathological effect as well as any possible interaction with other microbial pathogens used for the control of this pest.

Human metapneumovirus: Characteristics, and therapeutic advances 10 years after its discovery

Human metapneumovirus (hMPV), described for the first time in 2001 by the team of Pr Albert Osterhaus [1], is one of the main viral pathogens responsible for human respiratory tract infections. It may cause death in elderly and immunocompromised patients. The agent most closely related to hMPV is the human respiratory syncytial virus (hRSV), the main virus involved in bronchiolitis and pneumonia in young children and in respiratory tract infections in the elderly. hMPV is defined by two main viral groups,Aand B, and has generallya winter annual distribution in temperate countries. Because of the difficulty to culture hMPV in vitro, its diagnosis is generally achieved using RT-PCR. Like other Paramyxoviridae, hMPVhas a negative stranded RNA genome that includes eight genes coding for nine different proteins. Its genomic organization and the functions of its surface attachment and fusion glycoproteins are relatively similar to those of hRSV. Although many studies have tried to explain the viral cycle of hMPV, there are still many unanswered questions on the exact roles of the viral proteins in the attachment, fusion and replication of hMPV. There is no approved modality to combat hMPV infections for the moment. The majority of the treatments currently tested on hMPV have already demonstrated an activity againsthRSVinfections. Some innovative approaches based on RNAi nterference and on fusion inhibitors could be effective in hMPV disease. One of the biggest problems to develop an effective hMPV vaccine is the difficulty of inducing a durable immune response. Several strategies such as live attenuated viruses generated by reverse genetics or recombinant proteins have been tested in animals with encouraging results.

NTPase and 5′-RNA Triphosphatase Activities of Chikungunya Virus nsP2 Protein

Chikungunya virus (CHIKV) is an insect borne virus (genus: Alphavirus) which causes acute febrile illness in humans followed by a prolonged arthralgic disease that affects the joints of the extremities. Re-emergence of the virus in the form of outbreaks in last 6–7 years has posed a serious public health problem. CHIKV has a positive sense single stranded RNA genome of about 12,000 nt. Open reading frame 1 of the viral genome encodes a polyprotein precursor, nsP1234, which is processed further into different non structural proteins (nsP1, nsP2, nsP3 and nsP4). Sequence based analyses have shown helicase domain at the N-terminus and protease domain at C-terminus of nsP2. A detailed biochemical analysis of NTPase/RNA helicase and 5′-RNA phosphatase activities of recombinant CHIKV-nsP2T protein (containing conserved NTPase/helicase motifs in the N-terminus and partial papain like protease domain at the C-terminus) was carried out. The protein could hydrolyze all NTPs except dTTP and showed better efficiency for ATP, dATP, GTP and dGTP hydrolysis. ATP was the most preferred substrate by the enzyme. CHIKV-nsP2T also showed 5′-triphosphatase (RTPase) activity that specifically removes the γ-phosphate from the 5′ end of RNA. Both NTPase and RTPase activities of the protein were completely dependent on Mg2+ ions. RTPase activity was inhibited by ATP showing sharing of the binding motif by NTP and RNA. Both enzymatic activities were drastically reduced by mutations in the NTP binding motif (GKT) and co-factor, Mg2+ ion binding motif (DEXX) suggesting that they have a common catalytic site.

Inhibition of Hazara nairovirus replication by small interfering RNAs and their combination with ribavirin

BackgroundThe genus Nairovirus in the family Bunyaviridae contains 34 tick-borne viruses classified into seven serogroups. Hazara virus (HAZV) belongs to the Crimean-Congo hemorrhagic fever (CCHF) serogroup that also includes CCHF virus (CCHFV) a major pathogen for humans. HAZV is an interesting model to study CCHFV due to a close serological and phylogenetical relationship and a classification which allows handling in a BSL2 laboratory. Nairoviruses are characterized by a tripartite negative-sense single stranded RNA genome (named L, M and S segments) that encode the RNA polymerase, the Gn-Gc glycoproteins and the nucleoprotein (NP), respectively. Currently, there are neither vaccines nor effective therapies for the treatment of any bunyavirus infection in humans. In this study we report, for the first time, the use of RNA interference (RNAi) as an approach to inhibit nairovirus replication.ResultsChemically synthesized siRNAs were designed to target the mRNA produced by the three genomic segments. We first demonstrated that the siRNAs targeting the NP mRNA displayed a stronger antiviral effect than those complementary to the L and M transcripts in A549 cells. We further characterized the two most efficient siRNAs showing, that the induced inhibition is specific and associated with a decrease in NP synthesis during HAZV infection. Furthermore, both siRNAs depicted an antiviral activity when used before and after HAZV infection. We next showed that HAZV was sensitive to ribavirin which is also known to inhibit CCHFV. Finally, we demonstrated the additive or synergistic antiviral effect of siRNAs used in combination with ribavirin.ConclusionsOur study highlights the interest of using RNAi (alone or in combination with ribavirin) to treat nairovirus infection. This approach has to be considered for the development of future antiviral compounds targeting CCHFV, the most pathogenic nairovirus.

Cyclophilin A Interacts with Domain II of Hepatitis C Virus NS5A and Stimulates RNA Binding in an Isomerase-Dependent Manner

NS5A plays a critical, yet poorly defined, role in hepatitis C virus genome replication. The protein consists of three domains, each of which is able to bind independently to the 3' untranslated region (UTR) of the viral positive strand genomic RNA. The peptidyl-prolyl isomerase cyclophilin A (CypA) binds to domain II, catalyzing cis-trans isomerization. CypA inhibitors such as cyclosporine (CsA) have been shown to inhibit hepatitis C virus (HCV) replication. We show here that CypA stimulated domain II RNA binding activity, and this stimulation was abrogated by CsA. An isomerase mutant of CypA (H126Q) failed to bind to domain II and did not stimulate RNA binding. Finally, we demonstrate that the RNA binding of two domain II mutants, the D316E and D316E/Y317N mutants, previously shown to exhibit CypA independence for RNA replication, was unaffected by CypA. This study provides an insight into the molecular mechanism of CypA activity during HCV replication and further validates the use of CypA inhibitors in HCV therapy.

Targeting HCV (+) Strand RNA Genome by a Novel PNA–neamine Conjugate

Targeting HCV (+) Strand RNA Genome by a Novel PNA–neamine Conjugate

A Comprehensive Deep Sequencing Strategy for Full-Length Genomes of Influenza A

Driven by the impact of influenza A viruses on human and animal health, much research is conducted on this pathogen. To support this research, we designed an all influenza A-embracing reverse transcription-PCR (RT-PCR) for the generation of DNA from influenza A virus negative strand RNA genome segments for full-length genome deep sequencing on a Genome Sequencer FLX instrument. For high reliability, the RT-PCRs are designed such that every genome segment is divided into two amplicons and for the most variable segments redundancy is included. Moreover, to minimize the risk of contamination of diagnostic real-time PCRs by sequencing amplicons, RT-PCR does not generate amplicons that are amenable to RT-qPCR detection. With the presented protocol we were able to generate virtually all amplicons (99.3% success rate) from isolates representing all so far known 16 hemagglutinin and 9 neuraminidase subtypes and from an additional 2009 pandemic influenza A H1N1 virus. Three isolates were sequenced to analyze the suitability of the DNA for sequencing. Moreover, we provide a short R script that disambiguates the sequences of the primers used. We show that using unambiguous primer sequences for read trimming prior to assembly with the genome sequencer assembler software results in higher quality of the final genome sequences. Using the disambiguated primer sequences, high quality full-length sequences for the three isolates used for sequencing trials could be established from the raw data in de novo assemblies.

Human MxA protein inhibits the replication of classical swine fever virus

Classical swine fever virus (CSFV) has a spherical enveloped particle with a single stranded RNA genome, the virus belonging to a pestivirus of the family Flaviviridae is the causative agent of an acute contagious disease classical swine fever (CSF). The interferon-induced MxA protein has been widely shown to inhibit the life cycle of certain RNA viruses as members of the Bunyaviridae family and others. Interestingly, it has been reported that expression of MxA in infected cells was blocked by CSFV and whether MxA has an inhibitory effect against CSFV remains unknown to date until present. Here, we report that CSFV replicated poorly in cells stably transfected with human MxA. The proliferation of progeny virus in both PK-15 cell lines and swine fetal fibroblasts (PEF) continuously expressing MxA was shown significantly inhibited as measured by virus titration, indirect immune fluorescence assay and real-time PCR.

A Sequence in Coat Protein Open Reading Frame Is Required for Turnip Yellow Mosaic Virus Replication

Turnip yellow mosaic virus (TYMV) is a spherical plant virus that has a single 6.3 kb positive strand RNA genome. Information for TYMV replication is limited, except that the 3’-terminal sequence and 5’-untranslated region are required for genome replication. When a foreign sequence was inserted at the position upstream of the coat protein (CP) open reading frame (ORF), replication of the recombinant TYMV was comparable to wild type, as long as an RNAi suppressor was provided. In contrast, when the foreign sequence was inserted between the CP ORF and the 3’-terminal tRNA-like structure, replication of the recombinant virus was not detected. This result suggests that the CP ORF contains an essential replication element which should be appropriately spaced with respect to the 3’-end. Analysis of TYMV constructs containing a part or a full additional CP ORF indicates that the 3’ quarter of the CP ORF is required for TYMV replication.

A balance between circular and linear forms of the dengue virus genome is crucial for viral replication

The plasticity of viral plus strand RNA genomes is fundamental for the multiple functions of these molecules. Local and long-range RNA-RNA interactions provide the scaffold for interacting proteins of the translation, replication, and encapsidation machinery. Using dengue virus as a model, we investigated the relevance of the interplay between two alternative conformations of the viral genome during replication. Flaviviruses require long-range RNA-RNA interactions and genome cyclization for RNA synthesis. Here, we define a sequence present in the viral 3'UTR that overlaps two mutually exclusive structures. This sequence can form an extended duplex by long-range 5'-3' interactions in the circular conformation of the RNA or fold locally into a small hairpin (sHP) in the linear form of the genome. A mutational analysis of the sHP structure revealed an absolute requirement of this element for viral viability, suggesting the need of a linear conformation of the genome. Viral RNA replication showed high vulnerability to changes that alter the balance between circular and linear forms of the RNA. Mutations that shift the equilibrium toward the circular or the linear conformation of the genome spontaneously revert to sequences with different mutations that tend to restore the relative stability of the two competing structures. We propose a model in which the viral genome exists in at least two alternative conformations and the balance between these two states is critical for infectivity.

Unexpected inheritance: multiple integrations of ancient bornavirus and ebolavirus/marburgvirus sequences in vertebrate genomes.

Vertebrate genomes contain numerous copies of retroviral sequences, acquired over the course of evolution. Until recently they were thought to be the only type of RNA viruses to be so represented, because integration of a DNA copy of their genome is required for their replication. In this study, an extensive sequence comparison was conducted in which 5,666 viral genes from all known non-retroviral families with single-stranded RNA genomes were matched against the germline genomes of 48 vertebrate species, to determine if such viruses could also contribute to the vertebrate genetic heritage. In 19 of the tested vertebrate species, we discovered as many as 80 high-confidence examples of genomic DNA sequences that appear to be derived, as long ago as 40 million years, from ancestral members of 4 currently circulating virus families with single strand RNA genomes. Surprisingly, almost all of the sequences are related to only two families in the Order Mononegavirales: the Bornaviruses and the Filoviruses, which cause lethal neurological disease and hemorrhagic fevers, respectively. Based on signature landmarks some, and perhaps all, of the endogenous virus-like DNA sequences appear to be LINE element-facilitated integrations derived from viral mRNAs. The integrations represent genes that encode viral nucleocapsid, RNA-dependent-RNA-polymerase, matrix and, possibly, glycoproteins. Integrations are generally limited to one or very few copies of a related viral gene per species, suggesting that once the initial germline integration was obtained (or selected), later integrations failed or provided little advantage to the host. The conservation of relatively long open reading frames for several of the endogenous sequences, the virus-like protein regions represented, and a potential correlation between their presence and a species' resistance to the diseases caused by these pathogens, are consistent with the notion that their products provide some important biological advantage to the species. In addition, the viruses could also benefit, as some resistant species (e.g. bats) may serve as natural reservoirs for their persistence and transmission. Given the stringent limitations imposed in this informatics search, the examples described here should be considered a low estimate of the number of such integration events that have persisted over evolutionary time scales. Clearly, the sources of genetic information in vertebrate genomes are much more diverse than previously suspected.