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α-galactosidase (α-GAL) and α-N-acetylgalactosaminidase (α-NAGAL) are two glycosyl hydrolases responsible for maintaining cellular homeostasis by regulating glycan substrates on proteins and lipids. Mutations in the human genes encoding either enzyme lead to neurological and neuromuscular impairments seen in both Fabry- and Schindler/Kanzaki- diseases. Here, we investigate whether the parasitic blood fluke Schistosoma mansoni, responsible for the neglected tropical disease schistosomiasis, also contains functionally important α-GAL and α-NAGAL proteins. As infection, parasite maturation and host interactions are all governed by carefully-regulated glycosylation processes, inhibiting S. mansoni’s α-GAL and α-NAGAL activities could lead to the development of novel chemotherapeutics. Sequence and phylogenetic analyses of putative α-GAL/α-NAGAL protein types showed Smp_089290 to be the only S. mansoni protein to contain the functional amino acid residues necessary for α-GAL/α-NAGAL substrate cleavage. Both α-GAL and α-NAGAL enzymatic activities were higher in females compared to males (p α-GAL), which was consistent with smp_089290’s female biased expression. Spatial localisation of smp_089290 revealed accumulation in parenchymal cells, neuronal cells, and the vitellaria and mature vitellocytes of the adult schistosome. siRNA-mediated knockdown (>90%) of smp_089290 in adult worms significantly inhibited α-NAGAL activity when compared to control worms (siLuc treated males, p, Author summary Schistosomiasis is a parasitic disease caused by infection with blood flukes, which leads to acute and chronic pathology in millions of infected individuals located in deprived tropical and subtropical regions. Elucidating the function of schistosome genes has provided a clearer view on their roles in various molecular pathways, which are critical to successful parasitism. This information is invaluable when progressing novel drug and vaccine candidates. Here, we add to the existing knowledge of the Schistosoma mansoni parasitic glycan processing and modification machinery by functionally characterising a glycosyl hydrolase (S. mansoni α-N-acetylgalactosaminidase, SmNAGAL). We demonstrate that this protein is enzymatically active and important in coordinating parasite movement in adult male and female schistosomes. Additionally, we provide evidence that this protein regulates pathways associated with egg production in female schistosomes, which is responsible for inducing pathological reactions. Developing drugs that inhibit SmNAGAL enzymatic activity could provide a novel approach for controlling schistosomiasis.

Citrus Huanglongbing (HLB), also known as citrus greening, is one of the most devastating citrus diseases worldwide. Candidatus Liberibacter asiaticus (CLas) is the most prevalent strain associated with HLB, which is yet to be cultured in vitro. None of the commercial citrus cultivars are resistant to HLB. The pathosystem of Ca. Liberibacter is complex and remains a mystery. In this review, we focus on the recent progress in genomic research on the pathogen, the interaction of host and CLas, and the influence of CLas infection on the transcripts, proteins, and metabolism of the host. We have also focused on the identification of candidate genes for CLas pathogenicity or the improvements of HLB tolerance in citrus. In the end, we propose potentially promising areas for mechanistic studies of CLas pathogenicity, defense regulators, and genetic improvement for HLB tolerance/resistance in the future.

Schistosomes infect over 200 million of the world’s poorest people, but unfortunately treatment relies on a single drug. Nuclear hormone receptors are ligand-activated transcription factors that regulate diverse processes in metazoans, yet few have been functionally characterized in schistosomes. During a systematic analysis of nuclear receptor function, we found that an FTZ-F1-like receptor was essential for parasite survival. Using a combination of transcriptional profiling and chromatin immunoprecipitation (ChIP), we discovered that the micro-exon gene meg-8.3 is a transcriptional target of SmFTZ-F1. We found that both Smftz-f1 and meg-8.3 are required for esophageal gland maintenance as well as integrity of the worm’s head. Together, these studies define a new role for micro-exon gene function in the parasite and suggest that factors associated with the esophageal gland could represent viable therapeutic targets., Author summary Schistosomes kill an estimated 250,000 people every year and cause severe morbidity in millions more. Therefore, it is critical we continue to understand fundamental processes in the parasite so new therapies can be developed. Here, we identify a transcriptional regulator protein SmFTZ-F1 that is critical for parasite survival. We find that this protein controls the levels of a schistosome-specific gene (meg-8.3) and that meg-8.3 is critical for the maintenance of the worm’s esophageal gland. Loss of \ either meg-8.3 or Smftz-f1 leads to degeneration of the parasites’ head tissues. These studies suggest that molecules associated with esophageal gland function could represent therapeutic targets.

Our knowledge of citrus viruses is largely skewed toward virus pathology in cultivated orchards. Little is known about the virus diversity in wild citrus species. Here, we used a metatranscriptomics approach to characterize the virus diversity in a wild citrus habitat within the proposed center of the origin of citrus plants. We discovered a total of 44 virus isolates that could be classified into species Citrus tristeza virus and putative species citrus associated ampelovirus 1, citrus associated ampelovirus 2, and citrus virus B within the family Closteroviridae, providing important information to explore the factors facilitating outbreaks of citrus viruses and the evolutionary history of the family Closteroviridae. We found that frequent horizontal gene transfer, gene duplication, and alteration of expression strategy have shaped the genome complexity and diversification of the family Closteroviridae. Recombination frequently occurred among distinct Closteroviridae members, thereby facilitating the evolution of Closteroviridae. Given the potential emergence of similar wild-citrus-originated novel viruses as pathogens, the need for surveillance of their pathogenic and epidemiological characteristics is of utmost priority for global citrus production., Author summary Closterovirids are principal plant pathogens for citrus trees and other plants, as they sometimes cause new or re-emerging diseases. However, the closterovirid diversity in natural plant hosts, especially citrus plants, is unclear. Here, we describe three novel species and Citrus tristeza virus within the family Closteroviridae that were sampled from wild citrus trees growing in their natural habitat in southwestern China. The presence of three different taxon classes of the family Closteroviridae indicates the geographical uniqueness of the sampling region for citrus closterovirid evolution. Our analysis shows that frequent horizontal gene transfer, gene duplication, alteration of expression strategy, and recombination have been important evolutionary processes in the diversification of the family Closteroviridae. Our study also shows the significance of natural reserves as potential sources of disease agents endangering cultivated crop plants.

Animal-parasitic nematodes have thus far been largely refractory to genetic manipulation, and methods employed to effect RNA interference (RNAi) have been ineffective or inconsistent in most cases. We describe here a new approach for genetic manipulation of Nippostrongylus brasiliensis, a widely used laboratory model of gastrointestinal nematode infection. N. brasiliensis was successfully transduced with Vesicular Stomatitis Virus glycoprotein G (VSV-G)-pseudotyped lentivirus. The virus was taken up via the nematode intestine, RNA reverse transcribed into proviral DNA, and transgene transcripts produced stably in infective larvae, which resulted in expression of the reporter protein mCherry. Improved transgene expression was achieved by incorporating the C. elegans hlh11 promoter and the tbb2 3´-UTR into viral constructs. MicroRNA-adapted short hairpin RNAs delivered in this manner were processed correctly and resulted in partial knockdown of β-tubulin isotype-1 (tbb-iso-1) and secreted acetylcholinesterase B (ache-B). The system was further refined by lentiviral delivery of double stranded RNAs, which acted as a trigger for RNAi following processing and generation of 22G-RNAs. Virus-encoded sequences were detectable in F1 eggs and third stage larvae, demonstrating that proviral DNA entered the germline and was heritable. Lentiviral transduction thus provides a new means for genetic manipulation of parasitic nematodes, including gene silencing and expression of exogenous genes., Author summary The complex life cycle of parasitic nematodes makes them very difficult to manipulate genetically, and methods to delete or silence genes which are routinely used in other organisms are ineffective in most species of nematodes which infect animals. This has hindered attempts to understand the function of defined genes and proteins, and their roles in development and interaction of nematode parasites with their host. We show here that foreign genetic material can be introduced into a widely used laboratory model of intestinal nematode infection by using a viral vector. The vector was modified to improve transgene expression, and a reporter protein expressed by transduced nematode larvae in vitro. We subsequently utilised the viral vector to deliver double stranded RNA molecules to the larvae. These molecules were processed along known pathways, resulting in partial knockdown of two test genes. This system represents a new means of genetically manipulating nematode parasites, and will aid in understanding their complex biology, in addition to defining new targets for control of infection.

Insects are known to host a wide variety of beneficial microbes that are fundamental to many aspects of their biology and have substantially shaped their evolution. Notably, parasitoid wasps have repeatedly evolved beneficial associations with viruses that enable developing wasps to survive as parasites that feed from other insects. Ongoing genomic sequencing efforts have revealed that most of these virus-derived entities are fully integrated into the genomes of parasitoid wasp lineages, representing endogenous viral elements (EVEs) that retain the ability to produce virus or virus-like particles within wasp reproductive tissues. All documented parasitoid EVEs have undergone similar genomic rearrangements compared to their viral ancestors characterized by viral genes scattered across wasp genomes and specific viral gene losses. The recurrent presence of viral endogenization and genomic reorganization in beneficial virus systems identified to date suggest that these features are crucial to forming heritable alliances between parasitoid wasps and viruses. Here, our genomic characterization of a mutualistic poxvirus associated with the wasp Diachasmimorpha longicaudata, known as Diachasmimorpha longicaudata entomopoxvirus (DlEPV), has uncovered the first instance of beneficial virus evolution that does not conform to the genomic architecture shared by parasitoid EVEs with which it displays evolutionary convergence. Rather, DlEPV retains the exogenous viral genome of its poxvirus ancestor and the majority of conserved poxvirus core genes. Additional comparative analyses indicate that DlEPV is related to a fly pathogen and contains a novel gene expansion that may be adaptive to its symbiotic role. Finally, differential expression analysis during virus replication in wasps and fly hosts demonstrates a unique mechanism of functional partitioning that allows DlEPV to persist within and provide benefit to its parasitoid wasp host., Author summary Viruses have repeatedly formed long-term associations with insects called parasitoid wasps, which grow as parasites within other insect hosts. While these viruses were once pathogenic, they have since been co-opted by parasitoid wasps to benefit the survival of wasp offspring during parasitism. The genomes of most identified beneficial viruses are fully integrated into the genomes of the parasitoid wasps that produce them. Because these virus-derived entities have lost the ability to exist apart from their associated wasps, they are considered endogenous viral elements (EVEs) of the wasps rather than mutualistic symbionts. We sequenced the genome of the beneficial parasitoid virus Diachasmimorpha longicaudata entomopoxvirus (DlEPV) and found that its genome is not integrated into the genome of Diachasmimorpha longicaudata wasps and has largely retained the genomic structure of its pathogenic ancestor. Given the importance of viral genome integration in the overall stability of parasitoid wasp-EVE systems, we identified a novel strategy used by DlEPV to maintain its relationship with D. longicaudata despite its lack of endogenization. Our findings in this study demonstrate the first instance of a mutualistic viral symbiont in insects and provide new insight into the means through which beneficial viruses can arise.

Antibiotic treatment has emerged as a promising strategy to sterilize and kill filarial nematodes due to their dependence on their endosymbiotic bacteria, Wolbachia. Several studies have shown that novel and FDA-approved antibiotics are efficacious at depleting the filarial nematodes of their endosymbiont, thus reducing female fecundity. However, it remains unclear if antibiotics can permanently deplete Wolbachia and cause sterility for the lifespan of the adult worms. Concerns about resistance arising from mass drug administration necessitate a careful exploration of potential Wolbachia recrudescence. In the present study, we investigated the long-term effects of the FDA-approved antibiotic, rifampicin, in the Brugia pahangi jird model of infection. Initially, rifampicin treatment depleted Wolbachia in adult worms and simultaneously impaired female worm fecundity. However, during an 8-month washout period, Wolbachia titers rebounded and embryogenesis returned to normal. Genome sequence analyses of Wolbachia revealed that despite the population bottleneck and recovery, no genetic changes occurred that could account for the rebound. Clusters of densely packed Wolbachia within the worm’s ovarian tissues were observed by confocal microscopy and remained in worms treated with rifampicin, suggesting that they may serve as privileged sites that allow Wolbachia to persist in worms while treated with antibiotic. To our knowledge, these clusters have not been previously described and may be the source of the Wolbachia rebound., Author summary Onchocerciasis (river blindness) and lymphatic filariasis (elephantiasis) are two neglected tropical diseases caused by filarial nematodes, which harbor the endosymbiotic bacteria, Wolbachia. Major efforts to discover new drugs to treat these diseases have led to the discovery of novel compounds including those that target Wolbachia. We investigated the long-term effects of rifampicin on the filarial nematode, Brugia pahangi, and its endosymbiont, Wolbachia, in an in vivo rodent model of infection. Initially, Wolbachia titers were significantly reduced by 95% and female fecundity was impaired shortly after treatment. 8 months later however, Wolbachia rebounded and embryogenesis returned to normal. Sequence analysis of the Wolbachia genome revealed that despite the population bottleneck and recovery, no genetic changes occurred that could account for the rebound. Clusters of Wolbachia were observed within the ovaries of female worms throughout the entire 8-month study. These clusters may sequester Wolbachia and allow the bacteria to persist during antibiotic treatment, thereby enabling them to repopulate ovarian tissues and ensuring their vertical transmission to future generations of microfilariae.

Wolbachia are maternally transmitted intracellular bacteria that induce a range of pathogenic and fitness-altering effects on insect and nematode hosts. In parasitoid wasps of the genus Trichogramma, Wolbachia infection induces asexual production of females, thus increasing transmission of Wolbachia. It has been hypothesized that Wolbachia infection accompanies a modification of the host epigenome. However, to date, data on genome-wide epigenomic changes associated with Wolbachia are limited, and are often confounded by background genetic differences. Here, we took sexually reproducing Trichogramma free of Wolbachia and introgressed their genome into a Wolbachia-infected cytoplasm, converting them to Wolbachia-mediated asexuality. Wolbachia was then cured from replicates of these introgressed lines, allowing us to examine the genome-wide effects of wasps newly converted to asexual reproduction while controlling for genetic background. We thus identified gene expression and DNA methylation changes associated with Wolbachia-infection. We found no overlaps between differentially expressed genes and differentially methylated genes, indicating that Wolbachia-infection associated DNA methylation change does not directly modulate levels of gene expression. Furthermore, genes affected by these mechanisms exhibit distinct evolutionary histories. Genes differentially methylated due to the infection tended to be evolutionarily conserved. In contrast, differentially expressed genes were significantly more likely to be unique to the Trichogramma lineage, suggesting host-specific transcriptomic responses to infection. Nevertheless, we identified several novel aspects of Wolbachia-associated DNA methylation changes. Differentially methylated genes included those involved in oocyte development and chromosome segregation. Interestingly, Wolbachia-infection was associated with higher levels of DNA methylation. Additionally, Wolbachia infection reduced overall variability in gene expression, even after accounting for the effect of DNA methylation. We also identified specific cases where alternative exon usage was associated with DNA methylation changes due to Wolbachia infection. These results begin to reveal distinct genes and molecular pathways subject to Wolbachia induced epigenetic modification and/or host responses to Wolbachia-infection., Author summary Wolbachia is an extremely common endosymbiotic infection of arthropods and nematodes. One of the reasons why Wolbachia can so successfully infect diverse species is the bacterium’s ability to profoundly alter the reproductive behavior of its host. It has been proposed that Wolbachia may modify host’s epigenetic programs to alter its reproductive behavior. However, it has been difficult to study how epigenetic programs change with Wolbachia infection, due to the confounding effects of genetic backgrounds. Here, we studied host transcriptome and epigenome changes associated with Wolbachia infection in a homogenous genetic background, by carrying out an innovative introgression scheme. By doing so, we show, for the first time, high-resolution molecular consequences of intracellular infection and offer insights into epigenetic and transcriptomic regulation of invertebrates.

DNA viruses can hijack and manipulate the host chromatin state to facilitate their infection. Multiple lines of evidences reveal that DNA virus infection results in the host chromatin relocation, yet there is little known about the effects of viral infection on the architecture of host chromatin. Here, a combination of epigenomic, transcriptomic and biochemical assays were conducted to investigate the temporal dynamics of chromatin accessibility in response to Bombyx mori nucleopolyhedrovirus (BmNPV) infection. The high-quality ATAC-seq data indicated that progressive chromatin remodeling took place following BmNPV infection. Viral infection resulted in a more open chromatin architecture, along with the marginalization of host genome and nucleosome disassembly. Moreover, our results revealed that chromatin accessibility in uninfected cells was regulated by euchromatic modifications, whereas the viral-induced highly accessible chromatin regions were originally associated with facultative heterochromatic modification. Overall, our findings illustrate for the first time the organization and accessibility of host chromatin in BmNPV-infected cells, which lay the foundation for future studies on epigenomic regulation mediated by DNA viruses., Author summary As a well-studied arthropod-specific double-stranded DNA virus, Bombyx mori nucleopolyhedrovirus (BmNPV) is a representative member of baculoviruses. BmNPV infection results in significant host chromatin marginalization, which has also been found in most DNA viruses. However, the effects of baculovirus infection on the organization and accessibility of host chromatin are poorly understood. Here, by using ATAC-seq, we show that DNA virus BmNPV infection gradually remodels the accessibility of host chromatin. ATAC-seq data reveal that the marginalized host chromatin is a more accessible architecture along with the depletion of multi-nucleosome depositions. Moreover, our findings suggest the increased accessibility regions are regulated by the facultative heterochromatic modification. Overall, we provide a novel understanding of molecular mechanisms by which baculovirus and DNA viruses alter the organization of host chromatin in epigenomic regulation.

Bats are newly identified reservoirs of hantaviruses (HVs) among which very divergent HVs have been discovered in recent years. However, their significance for public health remains unclear since their seroprevalence as well as antigenic relationship with human-infecting HVs have not been investigated. In the present study archived tissues of 1,419 bats of 22 species from 6 families collected in 5 south and southwest provinces in China were screened by pan-HV RT-PCR following viral metagenomic analysis. As a result nine HVs have been identified in two bat species in two provinces and phylogenetically classified into two species, Laibin virus (LAIV, ICTV approved species, 1 strain) and Xuan son virus (XSV, proposed species, 8 strains). Additionally, 709 serum samples of these bats were also analyzed by ELISA to investigate the seroprevalence and cross-reactivity between different HVs using expressed recombinant nucleocapsid proteins (rNPs) of LAIV, XSV and Seoul virus (SEOV). The cross-reactivity of some bat sera were further confirmed by western blot (WB) using three rNPs followed by fluorescent antibody virus neutralization test (FAVNT) against live SEOV. Results showed that the total HV seropositive rate of bat sera was 18.5% (131/709) with many cross reacting with two or all three rNPs and several able to neutralize SEOV. WB analysis using the three rNPs and their specific hyperimmune sera demonstrated cross-reactivity between XSV/SEOV and LAIV/XSV, but not LAIV/SEOV, indicating that XSV is antigenically closer to human-infecting HVs. In addition a study of the distribution of the viruses identified an area covering the region between Chinese Guangxi and North Vietnam, in which XSV and LAIV circulate within different bat colonies with a high seroprevalence. A circulation sphere of bat-borne HVs has therefore been proposed., Author summary Some HVs are life-threatening pathogens predominantly carried and transmitted by rodents. In recent years bat-borne HVs have been identified in a broad range of bat species. To understand their significance to public health the present study conducted extensive investigations on genetic diversity, seroprevalence, distribution and cross antigenicity of bat-borne HVs in south and southwest China. The results provide the first profiling of cross-reactivity between bat-borne and human-infecting HVs, demonstrating that some bat sera can neutralize SEOV in cell culture. They also revealed that divergent bat-borne HVs co-exist and are widely distributed in Chinese Guangxi/Yunnan as well as in north Vietnam, resulting in identification of an area between China and Vietnam in which natural circulation of bat-borne HVs is maintained. Given the existence of bat-borne HVs genetically and antigenically close to human-infecting HVs, the need for extensive future studies is emphasized in order to assess the potential risk of these viruses to public health.

The human zinc finger antiviral protein (ZAP) recognizes RNA by binding to CpG dinucleotides. Mammalian transcriptomes are CpG-poor, and ZAP may have evolved to exploit this feature to specifically target non-self viral RNA. Phylogenetic analyses reveal that ZAP and its paralogue PARP12 share an ancestral gene that arose prior to extensive eukaryote divergence, and the ZAP lineage diverged from the PARP12 lineage in tetrapods. Notably, the CpG content of modern eukaryote genomes varies widely, and ZAP-like genes arose subsequent to the emergence of CpG-suppression in vertebrates. Human PARP12 exhibited no antiviral activity against wild type and CpG-enriched HIV-1, but ZAP proteins from several tetrapods had antiviral activity when expressed in human cells. In some cases, ZAP antiviral activity required a TRIM25 protein from the same or related species, suggesting functional co-evolution of these genes. Indeed, a hypervariable sequence in the N-terminal domain of ZAP contributed to species-specific TRIM25 dependence in antiviral activity assays. Crosslinking immunoprecipitation coupled with RNA sequencing revealed that ZAP proteins from human, mouse, bat and alligator exhibit a high degree of CpG-specificity, while some avian ZAP proteins appear more promiscuous. Together, these data suggest that the CpG- rich RNA directed antiviral activity of ZAP-related proteins arose in tetrapods, subsequent to the onset of CpG suppression in certain eukaryote lineages, with subsequent species-specific adaptation of cofactor requirements and RNA target specificity., Author summary To control viral infections, cells have evolved a variety of mechanisms that detect, modify and sometimes eliminate viral components. One of such mechanism is the Zinc Finger Antiviral Protein (ZAP) which binds RNA sequences that are rich in elements composed of a cytosine followed by a guanine. Selection of viral RNA can only be achieved because such elements are sparse in RNAs encoded by human genes. Here, we traced the molecular evolution of ZAP. We found that ZAP and a closely related gene, PARP12, originated from the same ancestral gene that existed in a predecessor of vertebrates and invertebrates. We found that ZAP proteins from mammals, birds and reptiles have antiviral activity but only in the presence of a co-factor, TRIM25, from the same species. ZAP proteins from birds were particularly interesting since they demonstrated a broader antiviral activity, primarily driven by relaxed requirement for cytosine-guanine. Our findings suggest that viruses that infect birds–which are important vectors for human diseases–are under differential selective pressures and this property may influence the outcome of interspecies transmission.

Comparative studies using non-parasitic model species such as Caenorhabditis elegans, have been very helpful in investigating the basic biology and evolution of parasitic nematodes. However, as phylogenetic distance increases, these comparisons become more difficult, particularly when outside of the nematode clade to which C. elegans belongs (V). One of the reasons C. elegans has nevertheless been used for these comparisons, is that closely related well characterized free-living species that can serve as models for parasites of interest are frequently not available. The Clade IV parasitic nematodes Strongyloides are of great research interest due to their life cycle and other unique biological features, as well as their medical and veterinary importance. Rhabditophanes, a closely related free-living genus, forms part of the Strongyloidoidea nematode superfamily. Rhabditophanes diutinus (= R. sp. KR3021) was included in the recent comparative genomic analysis of the Strongyloididae, providing some insight into the genomic nature of parasitism. However, very little is known about this species, limiting its usefulness as a research model. Here we provide a species description, name the species as R. diutinus and investigate its life cycle and subsequently gene expression in multiple life stages. We identified two previously unreported starvation induced life stages: dauer larvae and arrested J2 (J2A) larvae. The dauer larvae are morphologically similar to and are the same developmental stage as dauers in C. elegans and infective larvae in Strongyloides. As in C. elegans and Strongyloides, dauer formation is inhibited by treatment with dafachronic acid, indicating some genetic control mechanisms are conserved. Similarly, the expression patterns of putative dauer/infective larva control genes resemble each other, in particular between R. diutinus and Strongyloides spp. These findings illustrate and increase the usefulness of R. diutinus as a non-parasitic, easy to work with model species for the Strongyloididae for studying the evolution of parasitism as well as many aspects of the biology of Strongyloides spp, in particular the formation of infective larvae., Author summary Parasitic worms are an issue of great medical, veterinary, agricultural and economic importance, yet little is known about how worms become parasites. Comparative studies with non-parasitic model species like C. elegans have been useful, however, this usefulness decreases as the evolutionary distance between the species increases. One way to combat this is by having more well-studied closely related species to parasites of interest. To address this, we provide information about Rhabditophanes diutinus, a free-living nematode that is part of the same superfamily as the medically and veterinary important Strongyloides parasites. We provide analysis on its life cycle, in particular on two starvation induced life stages, along with gene expression data. Overall, this important information illustrates and improves the use of R. diutinus, as a non-parasitic model species for studying parasite evolution and basic biology within Strongyloides.

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Defective-Interfering RNAs (DI-RNAs) have long been known to play an important role in virus replication and transmission. DI-RNAs emerge during virus passaging in both cell-culture and their hosts as a result of non-homologous RNA recombination. However, the principles of DI-RNA emergence and their subsequent evolution have remained elusive. Using a combination of long- and short-read Next-Generation Sequencing, we have characterized the formation of DI-RNAs during serial passaging of Flock House virus (FHV) in cell-culture over a period of 30 days in order to elucidate the pathways and potential mechanisms of DI-RNA emergence and evolution. For short-read RNAseq, we employed ‘ClickSeq’ due to its ability to sensitively and confidently detect RNA recombination events with nucleotide resolution. In parallel, we used the Oxford Nanopore Technologies’s (ONT) MinION to resolve full-length defective and wild-type viral genomes. Together, these accurately resolve both rare and common RNA recombination events, determine the correlation between recombination events, and quantifies the relative abundance of different DI-RNAs throughout passaging. We observe the formation of a diverse pool of defective RNAs at each stage of viral passaging. However, many of these ‘intermediate’ species, while present in early stages of passaging, do not accumulate. After approximately 9 days of passaging we observe the rapid accumulation of DI-RNAs with a correlated reduction in specific infectivity and with the Nanopore data find that DI-RNAs are characterized by multiple RNA recombination events. This suggests that intermediate DI-RNA species are not competitive and that multiple recombination events interact epistatically to confer ‘mature’ DI-RNAs with their selective advantage allowing for their rapid accumulation. Alternatively, it is possible that mature DI-RNA species are generated in a single event involving multiple RNA rearrangements. These insights have important consequences for our understanding of the mechanisms, determinants and limitations in the emergence and evolution of DI-RNAs., Author summary Defective RNAs are versions of a viral genome that arise naturally during viral infections but have been truncated or rearranged by non-homologous recombination. While not encoding for functional viruses, they can be amplified and co-passaged with the wild-type virus, effectively parasitizing the normal viral machinery. Some defective RNAs can replicate so successfully so as to subdue the replication of the wild-type virus, forming a ‘Defective-Interfering RNA’ (DI-RNA). As a result, DI-RNAs may promote the establishment of chronic viral infections, may prolong the period during which the host is infectious, and may even be exploited as antiviral therapies or vaccines. Therefore, understanding the reasons and mechanisms of how DI-RNAs are formed and subsequently evolve is important. Here, we sought to characterize these processes by passaging Flock House virus (a highly tractable and well-characterized model RNA virus) in cell-culture for approximately one month and sequencing the viral genomes every three days using a combination of ‘ClickSeq’ (to resolve recombination events with nucleotide resolution) and the Oxford Nanopore Technologies MinION (to characterize full-length and defective genomes). This provides a highly detailed characterization of the pathways of DI-RNA emergence and their progression to dominance over the wild-type viral genome.

Schistosomiasis is the most important helminthic disease of humanity in terms of morbidity and mortality. Facile manipulation of schistosomes using lentiviruses would enable advances in functional genomics in these and related neglected tropical diseases pathogens including tapeworms, and including their non-dividing cells. Such approaches have hitherto been unavailable. Blood stream forms of the human blood fluke, Schistosoma mansoni, the causative agent of the hepatointestinal schistosomiasis, were infected with the human HIV-1 isolate NL4-3 pseudotyped with vesicular stomatitis virus glycoprotein. The appearance of strong stop and positive strand cDNAs indicated that virions fused to schistosome cells, the nucleocapsid internalized and the RNA genome reverse transcribed. Anchored PCR analysis, sequencing HIV-1-specific anchored Illumina libraries and Whole Genome Sequencing (WGS) of schistosomes confirmed chromosomal integration; >8,000 integrations were mapped, distributed throughout the eight pairs of chromosomes including the sex chromosomes. The rate of integrations in the genome exceeded five per 1,000 kb and HIV-1 integrated into protein-encoding loci and elsewhere with integration bias dissimilar to that of human T cells. We estimated ~ 2,100 integrations per schistosomulum based on WGS, i.e. about two or three events per cell, comparable to integration rates in human cells. Accomplishment in schistosomes of post-entry processes essential for HIV-1replication, including integrase-catalyzed integration, was remarkable given the phylogenetic distance between schistosomes and primates, the natural hosts of the genus Lentivirus. These enigmatic findings revealed that HIV-1 was active within cells of S. mansoni, and provided the first demonstration that HIV-1 can integrate into the genome of an invertebrate., Author Summary Schistosomiasis is a major neglected tropical disease (NTD), which afflicts > 200 million people in developing countries. The genome sequence of the schistosome parasite has been decoded; it includes > 10,000 genes. New approaches to control this NTD are sought and genomic information may provide targets for new treatments. Methods to determine the role and importance of specific genes would facilitate these tasks. The retrovirus HIV-1, the causative agent of HIV/AIDS, has been extensively studied and modified for use in biomedical research. Using a lab-modified form of HIV-1, we manipulated the genome of Schistosoma mansoni, one of the major species of schistosomes. Lab-modified HIV-1 infected schistosomes and inserted in the chromosomes of the parasite. These chromosomal insertions were mapped using next generation sequencing and were distributed throughout the chromosomes including the sex chromosomes. The findings were notable since they revealed that HIV-1 was active within cells of S. mansoni, and they provide the first demonstration that HIV-1 can integrate into the genome of an invertebrate. They pave a route forward for investigating new therapies for schistosomiasis.

Wolbachia are maternally transmitted intracellular bacterial symbionts that infect approximately 40% of all insect species. Though several strains of Wolbachia naturally infect Drosophila melanogaster and provide resistance against viral pathogens, or provision metabolites during periods of nutritional stress, one virulent strain, wMelPop, reduces fly lifespan by half, possibly as a consequence of over-replication. While the mechanisms that allow wMelPop to over-replicate are still of debate, a unique tandem repeat locus in the wMelPop genome that contains eight genes, referred to as the “Octomom” locus has been identified and is thought to play an important regulatory role. Estimates of Octomom locus copy number correlated increasing copy number to both Wolbachia bacterial density and increased pathology. Here we demonstrate that infected fly pathology is not dependent on an increased Octomom copy number, but does strongly correlate with increasing temperature. When measured across developmental time, we also show Octomom copy number to be highly variable across developmental time within a single generation. Using a second pathogenic strain of Wolbachia, we further demonstrate reduced insect lifespan can occur independently of a high Octomom locus copy number. Taken together, this data demonstrates that the mechanism/s of wMelPop virulence is more complex than has been previously described., Author Summary Wolbachia are obligate intracellular, symbiotic bacteria that infect approximately 40% of insect species, as well as filarial nematodes, arachnids and terrestrial isopods. While the vast majority of Wolbachia strains impose few fitness costs to their host, one strain wMelPop is unique as it lacks the ability to regulate its growth, and as consequence can reduce host lifespan by half. The strength of pathology induced by wMelPop has been linked to either increased bacterial density or copy number of an eight gene tandem repeat region referred to as the “Octomom” locus. To date no study has determined the effect changes to temperature have on Octomom copy number or bacterial density. Here we demonstrate that while the Octomom locus is unstable within a single generation of its host, changes to Octomom copy number did not occur in response to temperature. Furthermore, Octomom copy number or bacterial density does not correlate to the strength of pathology. These results indicate that the underpinning genetics of pathology are unclear, and the mechanisms by pathology is induced are more complex than previously realised.

Vector mosquitoes are responsible for transmission of the majority of arthropod-borne (arbo-) viruses. Virus replication in these vectors needs to be sufficiently high to permit efficient virus transfer to vertebrate hosts. The mosquito immune response therefore is a key determinant for arbovirus transmission. Mosquito antiviral immunity is primarily mediated by the small interfering RNA pathway. Besides this well-established antiviral machinery, the PIWI-interacting RNA (piRNA) pathway processes viral RNA into piRNAs. In recent years, significant progress has been made in characterizing the biogenesis and function of these viral piRNAs. In this review, we discuss these developments, identify knowledge gaps, and suggest directions for future research.

Polyomaviruses are a family of DNA tumor viruses that are known to infect mammals and birds. To investigate the deeper evolutionary history of the family, we used a combination of viral metagenomics, bioinformatics, and structural modeling approaches to identify and characterize polyomavirus sequences associated with fish and arthropods. Analyses drawing upon the divergent new sequences indicate that polyomaviruses have been gradually co-evolving with their animal hosts for at least half a billion years. Phylogenetic analyses of individual polyomavirus genes suggest that some modern polyomavirus species arose after ancient recombination events involving distantly related polyomavirus lineages. The improved evolutionary model provides a useful platform for developing a more accurate taxonomic classification system for the viral family Polyomaviridae., Author Summary Polyomaviruses are a family of DNA-based viruses that are known to infect various terrestrial vertebrates, including humans. In this report, we describe our discovery of highly divergent polyomaviruses associated with various marine fish. Searches of public deep sequencing databases unexpectedly revealed the existence of polyomavirus-like sequences in scorpion and spider datasets. Our analysis of these new sequences suggests that polyomaviruses have slowly co-evolved with individual host animal lineages through an established mechanism known as intrahost divergence. The proposed model is similar to the mechanisms through with other DNA viruses, such as papillomaviruses, are thought to have evolved. Our analysis also suggests that distantly related polyomaviruses sometimes recombine to produce new chimeric lineages. We propose a possible taxonomic scheme that can account for these inferred ancient recombination events.