Shrimp draft genome contains independent clusters of ancient and current endogenous viral elements (EVE) of the parvovirus IHHNV

Shrimp have the ability to accommodate viruses in long term, persistent infections without signs of disease. Endogenous viral elements (EVE) play a role in this process probably via production of negative-sense Piwi-interacting RNA (piRNA)-like fragments. These bind with Piwi proteins to dampen viral replication via the RNA interference (RNAi) pathway. We searched a draft genome of the giant tiger shrimp (Penaeus monodon)(GenBank record JABERT000000000) for the presence of EVE related to a shrimp parvovirus originally named infectious hypodermal and hematopoietic necrosis virus (IHHNV). The shrimp draft genome contained 3 piRNA-like gene clusters containing scrambled IHHNV EVE. Two clusters were located distant from one another in linkage group 35 (LG35). Both LG35 clusters contained multiple DNA fragments with high homology (99%) to GenBank records DQ228358 and EU675312 that were both called non-infectious IHHNV Type A (IHHNV-A) when originally discovered. However, our results and those from a recent Australian P. monodon genome assembly indicate that the relevant GenBank records for IHHNV-A are sequence-assembly artifacts derived from scrambled and fragmental IHHNV-EVE. Although the EVE in the two LG35 clusters showed high homology only to IHHNV-A, the clusters were separate and distinct with respect to the arrangement (i.e., order and reading direction) and proportional content of the IHHNV-A GenBank records. We conjecture that these 2 clusters may constitute independent allele-like clusters on a pair of homologous chromosomes. The third EVE cluster was found in linkage group 7 (LG7). It contained EVE with high homology (99%) only to GenBank record AF218266 with the potential to protect shrimp against infectious IHHNV. Our results suggested the possibility of viral-type specificity in EVE clusters. Specificity is important whole EVE clusters for one viral type would be transmitted to offspring as collective hereditary units. This would be advantageous if one or more of the EVE within the cluster were protective against disease caused by the cognate virus. It would also facilitate gene editing for removal of non-protective EVE clusters or for transfer of protective EVE clusters to genetically improve existing shrimp breeding stocks that might lack them.

A bioactive phlebovirus-like envelope protein in a hookworm endogenous virus

Endogenous viral elements (EVEs), accounting for 15% of our genome, serve as a genetic reservoir from which new genes can emerge. Nematode EVEs are particularly diverse and informative of virus evolution. We identify Atlas virus - an intact retrovirus-like EVE in the human hookworm Ancylostoma ceylanicum, with an envelope protein genetically related to GN-GC glycoproteins from phleboviruses. A cryo-EM structure of Atlas GC reveals a class II viral membrane fusion protein fold not previously seen in retroviruses. Atlas GC has the structural hallmarks of an active fusogen. Atlas GC trimers insert into membranes with endosomal lipid compositions and low pH. When expressed on the plasma membrane, Atlas GC has cell-cell fusion activity. RNA-Seq data analysis detected transcripts mapping to Atlas virus at different stages of hookworm development. With its preserved biological activities, Atlas GC has the potential to acquire a cellular function. Our work reveals structural plasticity in reverse-transcribing RNA viruses.

Genome assembly of the Australian black tiger shrimp (Penaeus monodon) reveals a fragmented IHHNV EVE sequence

Shrimp are a valuable aquaculture species globally; however, disease remains a major hindrance to shrimp aquaculture sustainability and growth. Mechanisms mediated by endogenous viral elements (EVEs) have been proposed as a means by which shrimp that encounter a new virus start to accommodate rather than succumb to infection over time. However, evidence on the nature of such EVEs and how they mediate viral accommodation is limited. More extensive genomic data on Penaeid shrimp from different geographical locations should assist in exposing the diversity of EVEs. In this context, reported here is a PacBio Sequel-based draft genome assembly of an Australian black tiger shrimp (Penaeus monodon) inbred for one generation. The 1.89 Gbp draft genome is comprised of 31,922 scaffolds (N50: 496,398 bp) covering 85.9% of the projected genome size. The genome repeat content (61.8% with 30% representing simple sequence repeats) is almost the highest identified for any species. The functional annotation identified 35,517 gene models, of which 25,809 were protein-coding and 17,158 were annotated using interproscan. Scaffold scanning for specific EVEs identified an element comprised of a 9,045 bp stretch of repeated, inverted and jumbled genome fragments of Infectious hypodermal and hematopoietic necrosis virus (IHHNV) bounded by a repeated 591/590 bp host sequence. As only near complete linear ~4 kb IHHNV genomes have been found integrated in the genome of P. monodon previously, its discovery has implications regarding the validity of PCR tests designed to specifically detect such linear EVE types. The existence of conjoined inverted IHHNV genome fragments also provides a means by which hairpin dsRNAs could be expressed and processed by the shrimp RNA interference (RNAi) machinery.

Extrachromosomal viral DNA produced by transcriptionally active endogenous viral elements in non-infected banana hybrids impedes quantitative PCR diagnostics of banana streak virus infections in banana hybrids

The main edible and cultivated banana varieties are intra- and interspecific hybrids of the two main Musa species, Musa acuminata and Musa balbisiana, having diploid genomes denoted A and B, respectively. The B genome naturally hosts sequences of banana streak virus (BSV) named endogenous BSV (eBSV). Upon stress, eBSVs are identified as the origin of BSV infection for at least three BSV species, causing banana streak disease. For each of the three species, BSV and eBSV share >99.9 % sequence identity, complicating PCR-based diagnosis of viral infection in the B genome-containing bananas. Here, we designed a quantitative PCR-based method to only quantify episomal BSV particles produced, overcoming the limitation of eBSV also being detected by qPCR by using it as a ‘calibrator’. However, our results revealed unexpected variation of eBSV amplification in calibrator plants composed of a clonal population of 53 replicating virus-free banana hybrids with the same AAB genotype. Our in-depth molecular analyses suggest that this calibrator variation is due to the variable abundance of non-encapsidated extrachromosomal viral DNA, likely produced via the transcription of eBSVs, followed by occasional reverse transcription. We also present evidence that accumulation of viral transcripts in AAB plants is downregulated both at post-transcriptional and transcriptional levels by an RNA interference mechanism that keeps the plants free of virus infection. Finally, we recommend that such eBSV amplification variation be taken into account to establish a quantitative viral diagnostic for banana plants with the B genome.

Shrimp Parvovirus Circular DNA Fragments Arise From Both Endogenous Viral Elements and the Infecting Virus

Some insects use endogenous reverse transcriptase (RT) to make variable viral copy DNA (vcDNA) fragments from viral RNA in linear (lvcDNA) and circular (cvcDNA) forms. The latter form is easy to extract selectively. The vcDNA produces small interfering RNA (siRNA) variants that inhibit viral replication via the RNA interference (RNAi) pathway. The vcDNA is also autonomously inserted into the host genome as endogenous viral elements (EVE) that can also result in RNAi. We hypothesized that similar mechanisms occurred in shrimp. We used the insect methods to extract circular viral copy DNA (cvcDNA) from the giant tiger shrimp (Penaeus monodon) infected with a virus originally named infectious hypodermal and hematopoietic necrosis virus (IHHNV). Simultaneous injection of the extracted cvcDNA plus IHHNV into whiteleg shrimp (Penaeus vannamei) resulted in a significant reduction in IHHNV replication when compared to shrimp injected with IHHNV only. Next generation sequencing (NGS) revealed that the extract contained a mixture of two general IHHNV-cvcDNA types. One showed 98 to 99% sequence identity to GenBank record AF218266 from an extant type of infectious IHHNV. The other type showed 98% sequence identity to GenBank record DQ228358, an EVE formerly called non-infectious IHHNV. The startling discovery that EVE could also give rise to cvcDNA revealed that cvcDNA provided an easy means to identify and characterize EVE in shrimp and perhaps other organisms. These studies open the way for identification, characterization and use of protective cvcDNA as a potential shrimp vaccine and as a tool to identify, characterize and select naturally protective EVE to improve shrimp tolerance to homologous viruses in breeding programs.

Genome-wide identification of endogenous viral sequences in alfalfa (Medicago sativa L.)

Endogenous viral elements (EVEs) have been for the most part described in animals and to a less extent in plants. The endogenization was proposed to contribute toward evolution of living organisms via horizontal gene transfer of novel genetic material and resultant genetic diversity. During the last two decades, several full-length and fragmented EVEs of pararetroviral and non-retroviral nature have been identified in different plant genomes, both monocots and eudicots. Prior to this work, no EVEs have been reported in alfalfa (Medicago sativa L.), the most cultivated forage legume in the world. In this study, taking advantage of the most recent developments in the field of alfalfa research, we have assessed alfalfa genome on the presence of viral-related sequences. Our analysis revealed segmented EVEs resembling two dsDNA reverse-transcribing virus species: Soybean chlorotic mottle virus (family Caulimoviridae, genus Soymovirus) and Figwort mosaic virus (family Caulimoviridae, genus Caulimovirus). The EVEs appear to be stable constituents of the host genome and in that capacity could potentially acquire functional roles in alfalfa’s development and response to environmental stresses.

Computational methods for the discovery and annotation of viral integrations

The transfer of genetic material between viruses and eukaryotic cells is pervasive. Somatic integrations of DNA viruses and retroviruses have been linked to persistent viral infection and genotoxic effects. Integrations into germline cells, referred to as Endogenous Viral Elements (EVEs), can be co-opted for host functions. Besides DNA viruses and retroviruses, EVEs can also derive from nonretroviral RNA viruses, which have often been observed in piRNA clusters. Here, we describe a bioinformatic framework to annotate EVEs in a genome assembly, study their widespread occurrence and polymorphism and identify sample-specific viral integrations using whole-genome sequencing data.

Insertion of Badnaviral DNA in the Late Blight Resistance Gene (R1a) of Brinjal Eggplant (Solanum melongena)

Endogenous viral elements (EVEs) are widespread in plant genomes. They result from the random integration of viral sequences into host plant genomes by horizontal DNA transfer and have the potential to alter host gene expression. We performed a large-scale search for co-transcripts including caulimovirid and plant sequences in 1,678 plant and 230 algal species and characterized 50 co-transcripts in 45 distinct plant species belonging to lycophytes, ferns, gymnosperms and angiosperms. We found that insertion of badnavirus EVEs along with Ty-1 copia mobile elements occurred into a late blight resistance gene (R1) of brinjal eggplant (Solanum melongena) and wild relatives in genus Solanum and disrupted R1 orthologs. EVEs of two previously unreported badnaviruses were identified in the genome of S. melongena, whereas EVEs from an additional novel badnavirus were identified in the genome of S. aethiopicum, the cultivated scarlet eggplant. Insertion of these viruses in the ancestral lineages of the direct wild relatives of the eggplant would have occurred during the last 3 Myr, further supporting the distinctiveness of the group of the eggplant within the giant genus Solanum.