Viral Genomic DNA Research Articles

Microbial composition in the nose of children with and without viruses during asthma exacerbations.

Recent evidence suggests microbial composition of the respiratory tract can influence respiratory health and disease. We sought to further elucidate the role of microbial communities in the upper respiratory tract during acute respiratory viral infections and exacerbations of asthma in a pediatric population. We recruited 155 children, ages 4-18 years, during asthma exacerbations (AE) (n=97) or during cold symptoms (CS) without history of asthma (n=58) from the emergency department over 4 years. A nasopharyngeal swab was obtained to provide both RNA for virus identification by viral genome sequencing and DNA for microbial composition by 16S rRNA gene sequencing. LEfSe analysis was performed to determine differences in relative contribution between microbial communities within and between groups. Subjects with AE and viral infection were found to have more abundant communities of Pseudomonadales (Moraxellaceae) (LDA score 5; p<0.05), while those AE without virus were found to have more Bifidobacteriacea (LDA 3; p<0.05). Heat maps of bacterial communities clustered by weighted UniFrac suggest microbial composition initially separates between AE and CS cohorts. However, viral detection noticeably alters the microbial composition and further separates each cohort into those with and without infection. In our pediatric population, the bacterial family Moraxellaceae was most related to AE during viral infections. Furthermore, viral infection was associated with compositional shifts of the microbiota within each cohort (AE and CS). Taken together, our data suggests AEs are increased during a viral infection that happens concurrently with an upper-respiratory tract that is composed of specific bacterial communities, such as Moraxellaceae.

The Free Energy Landscape of Retroviral Integration and Molecular Mechanisms of DNA Compaction

Retroviral integration, the process of covalently inserting viral DNA into the host genome, is a point of no return in the retroviral replication cycle. Despite its importance, many mechanistic details remain poorly understood. Using prototype foamy virus (PFV) as a model system, we monitor target DNA binding, the ensuing strand transfer reaction, and final complex disassembly by AFM imaging and magnetic tweezers force spectroscopy [1]. Our results reveal how multivalent target interactions at discrete auxiliary interfaces of the intasome render target capture irreversible, while allowing dynamic site selection. Strand transfer results in allosteric conformational changes and an extremely stable strand transfer complex [1]. Building upon the results on the model system, we investigate human immunodeficiency virus (HIV) pre-integration complexes (PICs). HIV must enter the nucleus to integrate the viral DNA genome. Nuclear import is complicated by the size restriction imposed by nuclear pore complexes that require compaction of the viral DNA within PICs. While the precise protein composition and DNA folding mechanism within PICs are unknown, the viral enzyme integrase is a major constituent of PICs. Whereas the strand transfer reaction involves an integrase multimer assembled at the viral DNA ends, PICs contain 10-fold more integrase monomers than required for catalysis. We therefore speculate on a functional role for integrase beyond catalysis and investigate whether integrase could serve a structural role in mediating DNA compaction in PICs. We again use atomic force microscopy imaging and magnetic tweezers-based micromanipulation to demonstrate that HIV integrase induces flexible bends and oligomerizes on DNA into particles capable of dynamic bridging and looping, which allow for the conformational plasticity that provides a route towards efficient compaction of the viral genome. [1] Vanderlinden, et al., Nature Comm. (2019).

Vira-Ome: A Tool to Detect DNA Viruses in the Cell-Free DNA

Introduction Viral infections are commonly reported in immunocompromised patients such as hematopoietic stem cell transplant recipients. While there are several reports on the effect of individual viruses, such as CMV, there is increasing evidence that the composition of the blood-virome may reflect the immune status of patients. High resolution virome analysis may provide an accurate measure of a patient's immune status as well as provide pathogen surveillance. Next generation sequencing (NGS) of cell-free DNA (cfDNA) purified from plasma is being investigated for assessment of diseases such as graft vs host disease, making unbiased sequence data readily available for analyses of a patient's virome. Here we present a computational tool (Vira-ome) which probes the presence of a targeted panel of DNA viruses following NGS sequencing of cfDNA. Method We developed a pipeline which initially removes host DNA reads, then performs a reference-assisted assembly operation. The resulting assemblies were then passed to a K-mer profile based taxonomic classifier for annotation to the species level. We trained the K-mer based classifier for 3 different K-mer values using ∼14,000 DNA-virus genomes representing 36 prevalent and pathogenic species. Predictions of three different K-mer classifiers were used to make final predictions, applying a majority-wins rule. We tested our method on 30 simulated (1, 2, or 3 species mix with 50,100 or 500 NGS reads each) and 29 clinical samples obtained from a biorepository. Results The Vira-ome tool utilized cfDNA NGS data to screen for the presence of a targeted set of nine pathogenic viruses (human adenovirus, human herpesvirus 1, 2, 6, 7 and 8, BK polyomavirus, human parvovirus B19, JC polyomavirus, KI polyomavirus, WU polyomavirus, torque teno virus, Epstein Barr virus, human cytomegalovirus, and varicella-zoster virus). On the simulated set of viral sample mixes, our protocol had 100% accuracy with no false-positive predictions. For 29 clinical samples, our pipeline predicted: (A) Torque teno virus in 13/29 samples, (B) pathogenic viruses in 11/15 cases were predominated by BK polyomavirus (7/15 samples) and (C) at least one viral species in 10/14 cases. Selected samples were then analyzed by qPCR, which confirmed the presence of BKV, JCV, HHV7 and EBV in 8/9, 3/4, 1/1, 2/2 samples, respectively. Viral loads ranged from 6 – 106 copies/mL. Five samples predicted positive for the adenovirus, were not confirmed by qPCR, emphasizing the need of in vitro validation for in silico predictions. Conclusions The Vira-ome tool, designed to detect pathogenic viruses using cfDNA data, performed well both on simulated and clinical samples with a majority of results confirmed by qPCR. Our results emphasize how computational predictions can complement clinical diagnostic approaches.

Investigating the etiologic agents of aseptic meningitis outbreak in Iranian children.

Introduction:This study aimed to determine the viral agent (s) and their genome burden associated with an aseptic meningitis outbreak that occurred in Yasuj, Iran.Materials and Methods:During April to August 2015, 104 CSF samples from 104 patients under 14 years old admitted to the hospital of Yasuj, Iran, with aseptic meningitis associated clinical signs were collected. 200 μl CSF specimens was prepared for DNA and RNA viral genome extraction each and then subjected to diagnostic Taq-man real time PCR assays for the present of Enteroviruses, HSV, VZV, mumps, measles and rubella in the samples.Results:The majority of them had experienced clinical meningitis sign. Primary laboratory differentiated tests were in favor of viral meningitis. Among a total of 104 patients diagnosed with clinically aseptic meningitis, enterovirus as the most significant viral agent was detected in 53 subjects. However, mumps, HSV and VZV, as the endemic causes of viral meningitis, were detected in 6, 6 and 2 of the affected patients. It was revealed that two HSV and one VZV affected patients were co-infected with enteroviruses. All affected children with relatively variable viral load recovered without any sequels.Conclusion:The present study revealed enterovirus as the main predominant cause of pediatric aseptic meningitis that broke out in Yasuj-Iran. Also, the co-circulation of mumps, HSV and VZV, as the endemic cause during the same aseptic meningitis outbreak, was demonstrated in some cases.

Virus Latency and the Impact on Plants.

Plant viruses are thought to be essentially harmful to the lives of their cultivated crop hosts. In most cases studied, the interaction between viruses and cultivated crop plants negatively affects host morphology and physiology, thereby resulting in disease. Native wild/non-cultivated plants are often latently infected with viruses without any clear symptoms. Although seemingly non-harmful, these viruses pose a threat to cultivated crops because they can be transmitted by vectors and cause disease. Reports are accumulating on infections with latent plant viruses that do not cause disease but rather seem to be beneficial to the lives of wild host plants. In a few cases, viral latency involves the integration of full-length genome copies into the host genome that, in response to environmental stress or during certain developmental stages of host plants, can become activated to generate and replicate episomal copies, a transition from latency to reactivation and causation of disease development. The interaction between viruses and host plants may also lead to the integration of partial-length segments of viral DNA genomes or copy DNA of viral RNA genome sequences into the host genome. Transcripts derived from such integrated viral elements (EVEs) may be beneficial to host plants, for example, by conferring levels of virus resistance and/or causing persistence/latency of viral infections. Studies on viral latency in wild host plants might help us to understand and elucidate the underlying mechanisms of latency and provide insights into the raison d’être for viruses in the lives of plants.

Herpesviral lytic gene functions render the viral genome susceptible to novel editing by CRISPR/Cas9.

Herpes simplex virus (HSV) establishes lifelong latent infection and can cause serious human disease, but current antiviral therapies target lytic but not latent infection. We screened for sgRNAs that cleave HSV-1 DNA sequences efficiently in vitro and used these sgRNAs to observe the first editing of quiescent HSV-1 DNA. The sgRNAs targeted lytic replicating viral DNA genomes more efficiently than quiescent genomes, consistent with the open structure of lytic chromatin. Editing of latent genomes caused short indels while editing of replicating genomes produced indels, linear molecules, and large genomic sequence loss around the gRNA target site. The HSV ICP0 protein and viral DNA replication increased the loss of DNA sequences around the gRNA target site. We conclude that HSV, by promoting open chromatin needed for viral gene expression and by inhibiting the DNA damage response, makes the genome vulnerable to a novel form of editing by CRISPR-Cas9 during lytic replication.

Insights into Innate Sensing of Prototype Foamy Viruses in Myeloid Cells.

Foamy viruses (FVs) belong to the Spumaretrovirinae subfamily of retroviruses and are characterized by unique features in their replication strategy. This includes a reverse transcription (RTr) step of the packaged RNA genome late in replication, resulting in the release of particles with a fraction of them already containing an infectious viral DNA (vDNA) genome. Little is known about the immune responses against FVs in their hosts, which control infection and may be responsible for their apparent apathogenic nature. We studied the interaction of FVs with the innate immune system in myeloid cells, and characterized the viral pathogen-associated molecular patterns (PAMPs) and the cellular pattern recognition receptors and sensing pathways involved. Upon cytoplasmic access, full-length but not minimal vector genome containing FVs with active reverse transcriptase, induced an efficient innate immune response in various myeloid cells. It was dependent on cellular cGAS and STING and largely unaffected by RTr inhibition during viral entry. This suggests that RTr products, which are generated during FV morphogenesis in infected cells, and are therefore already present in FV particles taken up by immune cells, are the main PAMPs of FVs with full-length genomes sensed in a cGAS and STING-dependent manner by the innate immune system in host cells of the myeloid lineage.

Bmserpin2 Is Involved in BmNPV Infection by Suppressing Melanization in Bombyx mori.

Melanization, an important defense response, plays a vital role in arthropod immunity. It is mediated by serine proteases (SPs) that convert the inactive prophenoloxidase (PPO) to active phenoloxidase (PO) and is tightly regulated by serine protease inhibitors (serpins) which belong to a well distributed superfamily in invertebrates, participating in immune mechanisms and other important physiological processes. Here, we investigated the Bmserpin2 gene which was identified from a transcriptome database in response to Bombyx mori nucleopolyhedrovirus (BmNPV) infection. Quantitative real-time polymerase chain reaction (qRT-PCR) results showed that Bmserpin2 was expressed in all tissues, with maximum expression in fat body. Upon BmNPV infection, the expression of Bmserpin2 was up-regulated in P50 (susceptible strain) and BC9 (resistant strain) in haemocytes, fat body and the midgut. However, up-regulation was delayed in BC9 (48 or 72 h), in contrast to P50 (24 h), after BmNPV infection. Meanwhile, Bmserpin2 could delay or inhibit melanization in silkworm haemolymph. Significant increased PO activity can be observed in Bmserpin2-depleted haemolymph under NPV infection. Furthermore, the viral genomic DNA copy number was decreased in Bmserpin2-depleted haemolymph. We conclude that Bmserpin2 is an inducible gene which might be involved in the regulation of PPO activation and suppressed melanization, and have a potential role in the innate immune system of B. mori.

Quantification of plasma BK polyomavirus loads is affected by sequence variability, amplicon length, and non-encapsidated viral DNA genome fragments

Quantification of plasma BK polyomavirus loads is affected by sequence variability, amplicon length, and non-encapsidated viral DNA genome fragments

Duckling short beak and dwarfism syndrome virus infection activates host innate immune response involving both DNA and RNA sensors

Duckling short beak and dwarfism syndrome virus (SBDSV), a newly identified goose parvovirus, causes devastating disease in domestic waterfowl and considerable economic losses to Chinese waterfowl industry. The molecular pathogenesis of SBDSV infection, nature and dynamics of host immune responses against SBDSV infection remained elusive. In this study, we systematically explored the relative mRNA expression profiles of major innate immune-related genes in SBDSV infected duck embryo fibroblasts. We found that SBDSV infection effectively activated host innate immune responses and resulted in significant up-regulation of IFN-β and several vital IFN-stimulated genes (ISGs). These up-regulation responses were mainly attributed to viral genomic DNA and dsRNA replication intermediates. Importantly, the expression of cGAS was significantly induced, whereas the expression of other DNA receptors including DDX41, STING, ZBP1, LSM14A and LRRFIP1 have no significant change. Furthermore, SBDSV infection also activates the up-regulation of TLR3 and inhibited the expression of TLR2 and TLR4; however, no effect was observed on the expression of TLR1, TLR5, TLR7, TLR15 and TLR21. Intriguingly, SBDSV infection significantly up-regulated the expression of RNA sensors such as MDA5 and LGP2, and resulted in a delayed but significant up-regulation of RIG-I gene. Taken together, these data indicate that host multiple sensors including DNA sensor (cGAS) and RNA sensors (TLR3, MDA5 and LGP2) are involved in recognizing a variety of different pathogen associated molecular patterns (PAMPs) including viral genomic ssDNA and dsRNA replication intermediates, which trigger an effective antiviral innate immune response.

Oligonucleotide Enrichment of HSV-1 Genomic DNA from Clinical Specimens for Use in High-Throughput Sequencing.

To date more than 400 genomes of herpes simplex virus 1 (HSV-1) and the distantly related HSV-2 have been examined using deep sequencing techniques. This powerful approach has been especially useful for revealing the global genetic diversity that exists within and between strains of each virus species. However, most early methods for high-throughput sequencing required the input of abundant viral genomic DNA to enable the successful production of sequencing libraries, and the generation of sufficient short-read sequencing data for de novo genome assembly and similar applications. Therefore, the majority of sequenced HSV strains have been cultured and expanded in vitro prior to genomic analysis, to facilitate isolation of sufficient viral DNA for sequencing-library preparation. Here, we describe an in-solution targeted enrichment procedure for isolating, enriching, and sequencing HSV genomic DNA directly from clinical specimens. When this enrichment technique is combined with traditional sequencing-library preparation procedures, the need for in vitro culturing, expansion, and purification of viral DNA is eliminated. Furthermore, enrichment reduces the large amount of nonviral DNA that is typically present in specimens obtained directly from natural infections, thereby increasing the likelihood of successful viral genome sequencing and assembly. We have used this approach to prepare viral DNA libraries from clinical specimens derived from skin swabs, saliva, blood, and similar sources. We then use these libraries for deep sequencing and successful de novo assembly of the ~152kb viral genomes, at coverage depths exceeding 100-1000×, for both HSV-1 and HSV-2.

A simple and efficient method for potential point-of-care diagnosis of human papillomavirus genotypes: combination of isothermal recombinase polymerase amplification with lateral flow dipstick and reverse dot blot.

Cervical cancer is the second most common cancer in the world's woman population with a high incidence in developing countries where diagnostic conditions for the cancer are poor. The main culprit causing the cancer is the human papillomavirus (HPV). HPV is divided into three major groups, i.e., high-risk (HR) group, probable high-risk (pHR) group, and low-risk (LR) group according to their potential of causing cervical cancer. Therefore, developing a sensitive, reliable, and cost-effective point-of-care diagnostic method for the virus genotypes in developing countries even worldwide is of high importance for the cancer prevention and control strategies. Here we present a combined method of isothermal recombinase polymerase amplification (RPA), lateral flow dipstick (LFD), and reverse dot blot (RDB), in quick point-of-care identification of HPV genotypes. The combined method is highly specific to HPV when the conserved L1 genes are used as targeted genes for amplification. The method can be used in identification of HPV genotypes at point-of-care within 1h with a sensitivity of low to 100fg of the virus genomic DNA. We have demonstrated that it is an excellent diagnostic point-of-care assay in monitoring the disease without time-consuming and expensive procedures and devices.

An Interaction with PARP-1 and Inhibition of Parylation Contribute to Attenuation of DNA Damage Signaling by the Adenovirus E4orf4 Protein.

The adenovirus (Ad) E4orf4 protein was reported to contribute to inhibition of ATM- and ATR-regulated DNA damage signaling during Ad infection and following treatment with DNA-damaging drugs. Inhibition of these pathways improved Ad replication, and when expressed alone, E4orf4 sensitized transformed cells to drug-induced toxicity. However, the mechanisms utilized were not identified. Here, we show that E4orf4 associates with the DNA damage sensor poly(ADP-ribose) polymerase 1 (PARP-1) and that the association requires PARP activity. During Ad infection, PARP is activated, but its activity is not required for recruitment of either E4orf4 or PARP-1 to virus replication centers, suggesting that their association occurs following recruitment. Inhibition of PARP-1 assists E4orf4 in reducing DNA damage signaling during infection, and E4orf4 attenuates virus- and DNA damage-induced parylation. Furthermore, E4orf4 reduces PARP-1 phosphorylation on serine residues, which likely contributes to PARP-1 inhibition as phosphorylation of this enzyme was reported to enhance its activity. PARP-1 inhibition is important to Ad infection since treatment with a PARP inhibitor enhances replication efficiency. When E4orf4 is expressed alone, it associates with poly(ADP-ribose) (PAR) chains and is recruited to DNA damage sites in a PARP-1-dependent manner. This recruitment is required for inhibition of drug-induced ATR signaling by E4orf4 and for E4orf4-induced cancer cell death. Thus, the results presented here demonstrate a novel mechanism by which E4orf4 targets and inhibits DNA damage signaling through an association with PARP-1 for the benefit of the virus and impacting E4orf4-induced cancer cell death.IMPORTANCE Replication intermediates and ends of viral DNA genomes can be recognized by the cellular DNA damage response (DDR) network as DNA damage whose repair may lead to inhibition of virus replication. Therefore, many viruses evolved mechanisms to inhibit the DDR network. We have previously shown that the adenovirus (Ad) E4orf4 protein inhibits DDR signaling, but the mechanisms were not identified. Here, we describe an association of E4orf4 with the DNA damage sensor poly(ADP-ribose) polymerase 1 (PARP-1). E4orf4 reduces phosphorylation of this enzyme and inhibits its activity. PARP-1 inhibition assists E4orf4 in reducing Ad-induced DDR signaling and improves the efficiency of virus replication. Furthermore, the ability of E4orf4, when expressed alone, to accumulate at DNA damage sites and to kill cancer cells is attenuated by chemical inhibition of PARP-1. Our results indicate that the E4orf4-PARP-1 interaction has an important role in Ad replication and in promotion of E4orf4-induced cancer-selective cell death.

Ac25 in Autographa californica multiple nucleopolyhedrovirus was crucial for progeny budded virion production

To analyze the effect of Ac25 on the proliferation of AcMNPV (Autographa californica multicapsid nucleopolyhedrovirus) progeny virus and its function in virogenic stroma. AcMNPV is a model of baculovirus and is the most widely studied baculovirus. Ac25, as a single-stranded DNA-binding protein, is involved in viral genomic DNA replication. Viral proliferation assay showed that AcMNPV progeny virus could not be produced when Ac25 was knocked out, which indicated it was crucial for BV production. Absolute quantitative PCR analysis indicated that Ac25 was able to promote replication of the AcMNPV genome in host Sf9 cells. It was also found that Ac25 could increase the transcription level of 38k and vp39 late expression genes, and inhibit host cell proliferation. Ac25 is highly accumulated in the nucleus and promotes progeny virus production by stimulating viral genome replication and up-regulating the expression of late genes. Two potential applications of vAc-Ac25-EGFP were proposed: an improved bac-to-bac eukaryotic protein expression systems and biopesticides.

A Comprehensive Superposition of Viral Polymerase Structures.

Nucleic acid polymerases are essential enzymes that replicate the genomes of both RNA and DNA viruses. These enzymes are generally encoded by viruses themselves so as to provide biochemical functions and control elements that differ from those of the host cell polymerases. The core active site structure used by all replicative polymerases is highly conserved and composed of two key aspartate residues from the conserved motifs A and C, but beyond this there is significant divergence among structures. These differences can make it difficult to select which portions of structures to align for comparisons, yet there are extended structural similarities within different groups of viral polymerases that should clearly be considered to generate optimal alignments. This manuscript describes a comprehensive structure-based superposition of every viral polymerase structure solved thus far based on an alignment-tree approach wherein aligned regions grow in complexity as similarity among polymerases increases. The result is a set of 646 structures that have been aligned into a single common orientation. This provides a convenient resource for directly comparing viral polymerases and illustrating structural conservation among them. It also sets the stage for detailed bioinformatics analysis to further assess common structural features. The full set of protein data bank (PDB) formatted files is publicly available via the Polymerase Structures community page at the Zenodo.org open data repository.

Sequence-specific nucleases as tools for enhancing disease resistance in crops

Genome editing technologies, such as CRISPR/Cas, have recently become valuable tools for plant reverse genetics as well as crop improvement, including enhancement of disease resistance. Targeting susceptibility (S) genes by genome editing has proven to be a viable strategy for generating resistance to both bacterial and fungal pathogens in various crops. Examples include generating loss-of-function mutations in promoter elements of the SWEET S genes, which are targeted by transcription activator-like effectors secreted by many phytopathogenic Xanthomonas bacteria, as well as in the conserved MLO locus that confers susceptibility to powdery mildew fungal pathogens in many monocots and dicots. In addition to genome editing applications, CRISPR/Cas systems can be used as means of defending plants against viruses via targeting viral genomic DNA or RNA. Genome editing is therefore a highly promising approach that enables engineering disease resistance to various plant pathogens directly in elite cultivar background in a highly precise manner. Unlike conventional crop breeding, genome editing approaches are not relying on lengthy and laborious crosses/back-crosses involving parental and progeny lines and can significantly shorten the breeding timeline. Taking into account the high potential of genome editing technologies for both basic and applied plant science, the recent decision of the European Court of Justice to define transgene-free genetically edited crops as GMOs is, clearly, a backward step for the EU.

Hitchhiking of Viral Genomes on Cellular Chromosomes.

Persistent viral infections require a host cell reservoir that maintains functional copies of the viral genome. To this end, several DNA viruses maintain their genomes as extrachromosomal DNA minichromosomes in actively dividing cells. These viruses typically encode a viral protein that binds specifically to viral DNA genomes and tethers them to host mitotic chromosomes, thus enabling the viral genomes to hitchhike or piggyback into daughter cells. Viruses that use this tethering mechanism include papillomaviruses and the gammaherpesviruses Epstein-Barr virus and Kaposi's sarcoma-associated herpesvirus. This review describes the advantages and consequences of persistent extrachromosomal viral genome replication.

HIV-1 DNA sequence diversity and evolution during acute subtype C infection

Little is known about the genotypic make-up of HIV-1 DNA genomes during the earliest stages of HIV-1 infection. Here, we use near-full-length, single genome next-generation sequencing to longitudinally genotype and quantify subtype C HIV-1 DNA in four women identified during acute HIV-1 infection in Durban, South Africa, through twice-weekly screening of high-risk participants. In contrast to chronically HIV-1-infected patients, we found that at the earliest phases of infection in these four participants, the majority of viral DNA genomes are intact, lack APOBEC-3G/F-associated hypermutations, have limited genome truncations, and over one year show little indication of cytotoxic T cell-driven immune selections. Viral sequence divergence during acute infection is predominantly fueled by single-base substitutions and is limited by treatment initiation during the earliest stages of disease. Our observations provide rare longitudinal insights of HIV-1 DNA sequence profiles during the first year of infection to inform future HIV cure research.

Pepper yellow leaf curl Aceh virus: a novel bipartite begomovirus isolated from chili pepper, tomato, and tobacco plants in Indonesia.

During 2017, leaf samples of chili pepper (Capsicum annuum), tomato (Solanum lycopersicum), and tobacco (Nicotiana tabacum) plants exhibiting yellowing and curling symptoms were collected from Aceh province, Indonesia. These samples were used to isolate and sequence viral genomic DNA. Six isolates with complete DNA-A and DNA-B sequences of begomovirus were obtained, all of which showed >99% sequence identity to the others. DNA-A sequences shared the highest nucleotide sequence identity (89.3%-89.7%) with monopartite pepper yellow leaf curl Indonesia virus 2 (PepYLCIV2) and the second-highest sequence identity (87.3%-87.4%) with bipartite pepper yellow leaf curl Indonesia virus (PepYLCIV). The DNA-B sequences shared the highest nucleotide sequence identity (95%-97.5%) with PepYLCIV. Results of recombination analysis indicated that the novel begomovirus was a recombinant. In accordance with the guidelines for begomovirus species demarcation, these isolates should be assigned to a new species, and we have proposed the name ''pepper yellow leaf curl Aceh virus'' (PepYLCAV) for this virus.

The Widespread Occurrence and Potential Biological Roles of Endogenous Viral Elements in Insect Genomes.

Modern genomic sequencing and bioinformatics approaches have detected numerous examples of DNA sequences derived from DNA and RNA virus genomes integrated into both vertebrate and insect genomes. Retroviruses encode RNA-dependent DNA polymerases (reverse transcriptases) and integrases that convert their RNA viral genomes into DNA proviruses and facilitate proviral DNA integration into the host genome. Surprisingly, DNA sequences derived from RNA viruses that do not encode these enzymes also occur in host genomes. Non-retroviral integrated RNA virus sequences (NIRVS) occur at relatively high frequency in the genomes of the arboviral vectors Aedes aegypti and Aedes albopictus, are not distributed randomly and possibly contribute to mosquito antiviral immunity, suggesting these mosquitoes could serve as a model system for unravelling the function of NIRVS. Here we address the following questions: What drives DNA synthesis from the genomes of non-retroviral RNA viruses? How does integration of virus cDNA into host DNA occur, and what is its biological function (if any)? We review current knowledge of viral integrations in insect genomes, hypothesize mechanisms of NIRVS formation and their potential impact on insect biology, particularly antiviral immunity, and suggest directions for future research.