Development of Rapid and Affordable Virus-Mimicking Artificial Positive Controls.

A major bottleneck in the development of detection assays is the availability of positive controls. Their acquisition can be problematic, their maintenance is expensive, and without them, assays cannot be validated. Herein, we present a novel strategy for the development of virus-mimicking artificial positive controls (ViMAPCs). The time between design and application is less than 5 days, unlike alternatives which normally take several weeks to obtain and implement. The ViMAPCs provide a realistic representation of natural infection unlike alternatives and allow for an effortless recognition of laboratory-based contamination. The feasibility and adaptability of the strategy was evaluated using several RNA and DNA plant viruses. ViMAPCs can be used in diagnostics laboratories but also in the monitoring of pathogen outbreaks where rapid response is of utmost importance.

Genomic analyses of a widespread blueberry virus in the United States.

Screening of blueberry accessions using high throughput sequencing revealed the presence of a new virus. Genomic structure and sequence are similar to that of nectarine stem pitting associated virus (NSPaV), a member of the genus Luteovirus, family Tombusviridae. The full genome of the new luteovirus, tentatively named blueberry virus L (BlVL), was characterized and analyzed. Similar to NSPaV, BlVL does not contain readily identifiable movement proteins in any of the seven isolates sequenced. More than 600 samples collected from five states were screened and 79% were found infected, making BlVL the most widespread blueberry virus in the United States.

Characterization of the first Rubus yellow net virus genome from blackberry.

Rubus yellow net virus (RYNV) is a badnavirus that infects Rubus spp. Mixed infections with black raspberry necrosis virus and raspberry leaf mottle virus cause raspberry mosaic, a disease that leads to significant losses and even plant death. RYNV has been reported in several European countries and the Americas yet there is substantial lack of knowledge, especially when it comes to virus diversity and the evolutionary forces that affect virus fitness outside its primary host, raspberry. Herein, we report the first RYNV episomal genome isolated from blackberry and this is the first report of the virus in Bosnia and Herzegovina. The isolate has five open reading frames (ORFs) and, when compared with other fully sequenced counterparts, showed 82-97% nucleotide pairwise identity. This communication adds to our limited knowledge on RYNV and addresses some of the gaps in RYNV genetics when it comes to the coding capacity of episomal isolates and the probability of the first fully sequenced isolate of the virus being integrated in the raspberry genome.

Genomes of Bacteriophages Belonging to the Orders Caudovirales and Petitvirales Identified in Fecal Samples from Pacific Flying Fox (Pteropus tonganus) from the Kingdom of Tonga.

Twenty-nine circular genomes of bacteriophages in the orders Caudovirales and Petitvirales were identified from fecal samples from Pacific flying foxes that were collected from their roosting sites on the Pacific Island of Tonga in 2014 and 2015. The vast majority are microviruses (n = 25), with 2 siphoviruses, 1 myovirus, and 1 podovirus.

Diverse single-stranded DNA viruses identified in New Zealand (Aotearoa) South Island robin (Petroica australis) fecal samples.

The South Island robin (Petroica australis) is a small passerine bird endemic to New Zealand (Aotearoa). Although its population has declined recently and it is considered 'at risk,' little research has been done to identify viruses in this species. This study aimed to survey the diversity of single-stranded DNA viruses associated with South Island robins in a small, isolated population on Nukuwaiata Island. In total, 108 DNA viruses were identified from pooled fecal samples collected from 38 individual robins sampled. These viruses belong to the Circoviridae (n = 10), Genomoviridae (n = 12), and Microviridae (n = 73) families. A number of genomes that belong to the phylum Cressdnaviricota but are otherwise unclassified (n = 13) were also identified. These results greatly expand the known viral diversity associated with South Island robins, and we identify a novel group of viruses most closely related genomoviruses.

The population structure of the secovirid lychnis mottle virus based on the RNA2 coding sequences.

Lychnis mottle virus (LycMoV), family Secoviridae, is one of several viruses recently detected in peony. Given the high prevalence of the virus in the more than 300 samples tested, the population structure of the virus was studied using 48 isolates representing at least 20 cultivars and collected from major producing and propagating states in the United States. The homogeneity of the United States population, based on data from the RNA2 coding region, along with phylogenetic analyses of all publicly available sequences point to the dissemination of the virus through propagation material rather that active vector-mediated transmission.

Unveiling Crucivirus Diversity by Mining Metagenomic Data.

The discovery of cruciviruses revealed the most explicit example of a common protein homologue between DNA and RNA viruses to date. Cruciviruses are a novel group of circular Rep-encoding single-stranded DNA (ssDNA) (CRESS-DNA) viruses that encode capsid proteins that are most closely related to those encoded by RNA viruses in the family Tombusviridae The apparent chimeric nature of the two core proteins encoded by crucivirus genomes suggests horizontal gene transfer of capsid genes between DNA and RNA viruses. Here, we identified and characterized 451 new crucivirus genomes and 10 capsid-encoding circular genetic elements through de novo assembly and mining of metagenomic data. These genomes are highly diverse, as demonstrated by sequence comparisons and phylogenetic analysis of subsets of the protein sequences they encode. Most of the variation is reflected in the replication-associated protein (Rep) sequences, and much of the sequence diversity appears to be due to recombination. Our results suggest that recombination tends to occur more frequently among groups of cruciviruses with relatively similar capsid proteins and that the exchange of Rep protein domains between cruciviruses is rarer than intergenic recombination. Additionally, we suggest members of the stramenopiles/alveolates/Rhizaria supergroup as possible crucivirus hosts. Altogether, we provide a comprehensive and descriptive characterization of cruciviruses.IMPORTANCE Viruses are the most abundant biological entities on Earth. In addition to their impact on animal and plant health, viruses have important roles in ecosystem dynamics as well as in the evolution of the biosphere. Circular Rep-encoding single-stranded (CRESS) DNA viruses are ubiquitous in nature, many are agriculturally important, and they appear to have multiple origins from prokaryotic plasmids. A subset of CRESS-DNA viruses, the cruciviruses, have homologues of capsid proteins encoded by RNA viruses. The genetic structure of cruciviruses attests to the transfer of capsid genes between disparate groups of viruses. However, the evolutionary history of cruciviruses is still unclear. By collecting and analyzing cruciviral sequence data, we provide a deeper insight into the evolutionary intricacies of cruciviruses. Our results reveal an unexpected diversity of this virus group, with frequent recombination as an important determinant of variability.

Diverse genomoviruses representing twenty-nine species identified associated with plants.

Genomoviruses (family Genomoviridae) are circular single-stranded DNA viruses that have been mainly identified through metagenomics studies in a wide variety of samples from various environments. Here, we describe 98 genomes of genomoviruses found associated with members of 19 plant families from Australia, Brazil, France, South Africa and the USA. These 98 genomoviruses represent 29 species, 26 of which are new, in the genera Gemykolovirus (n = 37), Gemyduguivirus (n = 9), Gemygorvirus (n = 8), Gemykroznavirus (n = 6), Gemycircularvirus (n = 21) and Gemykibivirus (n = 17).

The population structure of Rose rosette virus in the USA.

Rose rosette virus (RRV) (genus Emaravirus) is the causal agent of the homonymous disease, the most destructive malady of roses in the USA. Although the importance of the disease is recognized, little sequence information and no full genomes are available for RRV, a multi-segmented RNA virus. To better understand the population structure of the virus we implemented a Hi-Plex PCR amplicon high-throughput sequencing approach to sequence all 7 segments and to quantify polymorphisms in 91 RRV isolates collected from 16 states in the USA. Analysis revealed insertion/deletion (indel) polymorphisms primarily in the 5' and 3' non-coding, but also within coding regions, including some resulting in changes of protein length. Phylogenetic analysis showed little geographical structuring, suggesting that topography does not have a strong influence on virus evolution. Overall, the virus populations were homogeneous, possibly because of regular movement of plants, the recent emergence of RRV and/or because the virus is under strong purification selection to preserve its integrity and biological functions.

Diverse papillomaviruses identified in Weddell seals.

Papillomaviridae is a diverse family of circular, double-stranded DNA (dsDNA) viruses that infect a broad range of mammalian, avian and fish hosts. While papillomaviruses have been characterized most extensively in humans, the study of non-human papillomaviruses has contributed greatly to our understanding of their pathogenicity and evolution. Using high-throughput sequencing approaches, we identified 7 novel papillomaviruses from vaginal swabs collected from 81 adult female Weddell seals (Leptonychotes weddellii) in the Ross Sea of Antarctica between 2014-2017. These seven papillomavirus genomes were amplified from seven individual seals, and six of the seven genomes represented novel species with distinct evolutionary lineages. This highlights the diversity of papillomaviruses among the relatively small number of Weddell seal samples tested. Viruses associated with large vertebrates are poorly studied in Antarctica, and this study adds information about papillomaviruses associated with Weddell seals and contributes to our understanding of the evolutionary history of papillomaviruses.

Molecular characterization of faba bean necrotic yellows viruses in Tunisia.

Faba bean necrotic yellows virus (FBNYV) (genus Nanovirus; family Nanoviridae) has a genome comprising eight individually encapsidated circular single-stranded DNA components. It has frequently been found infecting faba bean (Vicia faba L.) and chickpea (Cicer arietinum L.) in association with satellite molecules (alphasatellites). Genome sequences of FBNYV from Azerbaijan, Egypt, Iran, Morocco, Spain and Syria have been determined previously and we now report the first five genome sequences of FBNYV and associated alphasatellites from faba bean sampled in Tunisia. In addition, we have determined the genome sequences of two additional FBNYV isolates from chickpea plants sampled in Syria and Iran. All individual FBNYV genome component sequences that were determined here share > 84% nucleotide sequence identity with FBNYV sequences available in public databases, with the DNA-M component displaying the highest degree of diversity. As with other studied nanoviruses, recombination and genome component reassortment occurs frequently both between FBNYV genomes and between genomes of nanoviruses belonging to other species.

Unique genome organization of non-mammalian papillomaviruses provides insights into the evolution of viral early proteins.

The family Papillomaviridae contains more than 320 papillomavirus types, with most having been identified as infecting skin and mucosal epithelium in mammalian hosts. To date, only nine non-mammalian papillomaviruses have been described from birds (n = 5), a fish (n = 1), a snake (n = 1), and turtles (n = 2). The identification of papillomaviruses in sauropsids and a sparid fish suggests that early ancestors of papillomaviruses were already infecting the earliest Euteleostomi. The Euteleostomi clade includes more than 90 per cent of the living vertebrate species, and progeny virus could have been passed on to all members of this clade, inhabiting virtually every habitat on the planet. As part of this study, we isolated a novel papillomavirus from a 16-year-old female Adélie penguin (Pygoscelis adeliae) from Cape Crozier, Ross Island (Antarctica). The new papillomavirus shares ∼64 per cent genome-wide identity to a previously described Adélie penguin papillomavirus. Phylogenetic analyses show that the non-mammalian viruses (expect the python, Morelia spilota, associated papillomavirus) cluster near the base of the papillomavirus evolutionary tree. A papillomavirus isolated from an avian host (Northern fulmar; Fulmarus glacialis), like the two turtle papillomaviruses, lacks a putative E9 protein that is found in all other avian papillomaviruses. Furthermore, the Northern fulmar papillomavirus has an E7 more similar to the mammalian viruses than the other avian papillomaviruses. Typical E6 proteins of mammalian papillomaviruses have two Zinc finger motifs, whereas the sauropsid papillomaviruses only have one such motif. Furthermore, this motif is absent in the fish papillomavirus. Thus, it is highly likely that the most recent common ancestor of the mammalian and sauropsid papillomaviruses had a single motif E6. It appears that a motif duplication resulted in mammalian papillomaviruses having a double Zinc finger motif in E6. We estimated the divergence time between Northern fulmar-associated papillomavirus and the other Sauropsid papillomaviruses be to around 250 million years ago, during the Paleozoic-Mesozoic transition and our analysis dates the root of the papillomavirus tree between 400 and 600 million years ago. Our analysis shows evidence for niche adaptation and that these non-mammalian viruses have highly divergent E6 and E7 proteins, providing insights into the evolution of the early viral (onco-)proteins.

Identification of a Nanovirus-Alphasatellite Complex in Sophora alopecuroides.

Viruses in the genus Nanovirus of the family Nanoviridae generally have eight individually encapsidated circular genome components and have been predominantly found infecting Fabaceae plants in Europe, Australia, Africa and Asia. For over a decade Sophora alopecuroides L. (Fabaceae) plants have been observed across Iran displaying dwarfing, yellowing, stunted leaves and yellow vein banding. Using a high-throughput sequencing approach, sequences were identified within one such plant that had similarities to nanovirus genome components. From this plant, the nanovirus-like molecules DNA-R (n=4), DNA-C (n=2), DNA-S (n=1), DNA-M (n=1), DNA-N (n=1), DNA-U1 (n=1), DNA-U2 (n=1) and DNA-U4 (n=1) were amplified, cloned and sequenced. Other than for the DNA-R, these components share less than 71% identity with those of other known nanoviruses. The four DNA-R molecules were highly diverse, sharing only 65-71% identity with each other and 64-86% identity with those of other nanoviruses. In the S. alopecuroides plant 14 molecules sharing 57.7-84.6% identity with previously determined sequences of nanovirus-associated alphasatellites were also identified. Given the research activity in the nanovirus field during the last five years coupled with high-throughput sequence technologies, many more diverse nanoviruses and nanovirus-associated satellites are likely to be identified.

Novel Single-Stranded DNA Virus Genomes Recovered from Chimpanzee Feces Sampled from the Mambilla Plateau in Nigeria.

Metagenomic approaches are rapidly expanding our knowledge of the diversity of viruses. In the fecal matter of Nigerian chimpanzees we recovered three gokushovirus genomes, one circular replication-associated protein encoding single-stranded DNA virus (CRESS), and a CRESS DNA molecule.

Identification of a polyomavirus in Weddell seal (Leptonychotes weddellii) from the Ross Sea (Antarctica).

Viruses are ubiquitous in nature, however, very few have been identified that are associated with Antarctic animals. Here we report the identification of a polyomavirus in the kidney tissue of a deceased Weddell seal from the Ross Sea, Antarctica. The circular genome (5186 nt) has typical features of polyomaviruses with a small and larger T-antigen open reading frames (ORFs) and three ORFs encoding VP1, VP2 and VP3 capsid proteins. The genome of the Weddell seal polyomavirus (WsPyV) shares 85.4% genome-wide pairwise identity with a polyomavirus identified in a California sea lion. To our knowledge WsPyV is the first viral genome identified in Antarctic pinnipeds and the third polyomavirus to be identified from an Antarctic animal, the other two being from Adélie penguin (Pygoscelis adeliae) and a sharp-spined notothen (Trematomus pennellii), both sampled in the Ross sea. The GenBank accession number: KX533457.

Comparative analysis of common regions found in babuviruses and alphasatellite molecules.

Viruses in the genus Babuvirus have multi-component ssDNA genomes and often associate with alphasatellite molecules containing two common motifs, a common-region stem-loop (CR-SL) involved in initiation of rolling-circle replication and a common-region major (CR-M) motif involved in secondary-strand synthesis. We compared known babuvirus genome components and alphasatellite CR-SL and CR-M sequences, defining five divergent CR-SL sequence classes. We identified iterated sequence elements in babuvirus genome components that have particularly conserved sequences and spatial arrangements between known babuviruses.

Geometric morphometrics and molecular systematics of Xanthocnemis sobrina (McLachlan, 1873) (Odonata: Coenagrionidae) and comparison to its congeners.

The taxonomy of the damselfly genus Xanthocnemis is revised, with particular focus on populations inhabiting the North Island of New Zealand. Earlier studies revealed two species: X. sobrina, restricted to cool, shaded streams in kauri forests and other forested areas, and X. zealandica, a common species throughout New Zealand except the Chatham and subantarctic islands. A field study encompassing aquatic habitats throughout the whole North Island was carried out to establish the relationship between morphological variation (body size and various morphological traits over the entire body) observed by previous researchers with ecological conditions and/or geographical location. The main aim was to propose reliable diagnostic features that could be used in future studies. Morphological and molecular variation was assessed. Morphological examination included assigning landmarks for all body parts corresponding to the external morphological features that are usually used in Odonata taxonomy. Molecular analysis targeted fragments of the 28S and 16S rRNA genes. Congruence was sought between both types of data, statistical support for two morphological types previously described as different species and a maximum likelihood phylogenetic tree in conjunction with a pairwise genetic distance matrix constructed from the DNA sequences obtained from the sampled specimens. Geometric morphometrics revealed statistically significant differentiation between specimens identified as X. zealandica and X. sobrina for four traits: (1) dorsal view of the head for both sexes as well as male appendages from (2) dorsal, (3) ventral and (4) lateral views. Wings appeared different when analysed for males only. Molecular analysis, however, grouped all specimens into a single undifferentiated cluster with very low mean pairwise distance (<0.01) between them showing almost no variation at the molecular level among the sampled populations on the North Island. Therefore, an additional analysis of the mitochondrial cytochrome c-oxidase I gene was carried out comparing randomly selected North Island specimens to Xanthocnemis specimens targeted in other molecular studies (Nolan et al. 2007, Amaya-Perilla et al. 2014). The analysis of the COI gene confirmed that all North and South Island isolates of Xanthocnemis cluster together in a well-supported clade with pairwise identity >96% and ~93% pairwise identity with X. tuanuii sequences obtained from the Chatham Island specimens. A careful investigation of the thin plate spline deformations generated for the geometric morphometric landmarks showed that the significant variations in the appendages of the Xanthocnemis specimens appeared to be the result of size, rather than shape, differences. Therefore, X. sobrina is proposed as a synonym of X. zealandica. Recently Amaya-Perilla et al. (2014) synonymised X. sinclairi with X. zealandica and confirmed the status of the Chatham Island X. tuanuii as a distinct species. It is therefore proposed that the genus Xanthocnemis consists of two species only: zealandica occurring all over the North, South and Stewart Islands, and tuanuii, endemic to Chatham and Pitt islands. Considering several statistical tests involving body measurements and ecological variables recorded during the field study, as well as various discussion points from similar studies of other species of Odonata, two alternative hypotheses are proposed for future testing. The first hypothesis synonymises X. sobrina with X. zealandica and suggests a possible explanation for the evolution of the two morphological traits that have previously been considered diagnostic for these species. The second hypothesis suggests that as typical X. sobrina were not sampled during this study this could represent a species that is now extinct, unless future studies prove it otherwise.

Circular replication-associated protein encoding DNA viruses identified in the faecal matter of various animals in New Zealand.

In recent years, innovations in molecular techniques and sequencing technologies have resulted in a rapid expansion in the number of known viral sequences, in particular those with circular replication-associated protein (Rep)-encoding single-stranded (CRESS) DNA genomes. CRESS DNA viruses are present in the virome of many ecosystems and are known to infect a wide range of organisms. A large number of the recently identified CRESS DNA viruses cannot be classified into any known viral families, indicating that the current view of CRESS DNA viral sequence space is greatly underestimated. Animal faecal matter has proven to be a particularly useful source for sampling CRESS DNA viruses in an ecosystem, as it is cost-effective and non-invasive. In this study a viral metagenomic approach was used to explore the diversity of CRESS DNA viruses present in the faeces of domesticated and wild animals in New Zealand. Thirty-eight complete CRESS DNA viral genomes and two circular molecules (that may be defective molecules or single components of multicomponent genomes) were identified from forty-nine individual animal faecal samples. Based on shared genome organisations and sequence similarities, eighteen of the isolates were classified as gemycircularviruses and twelve isolates were classified as smacoviruses. The remaining eight isolates lack significant sequence similarity with any members of known CRESS DNA virus groups. This research adds significantly to our knowledge of CRESS DNA viral diversity in New Zealand, emphasising the prevalence of CRESS DNA viruses in nature, and reinforcing the suggestion that a large proportion of CRESS DNA viruses are yet to be identified.

Begomovirus-Associated Satellite DNA Diversity Captured Through Vector-Enabled Metagenomic (VEM) Surveys Using Whiteflies (Aleyrodidae).

Monopartite begomoviruses (Geminiviridae), which are whitefly-transmitted single-stranded DNA viruses known for causing devastating crop diseases, are often associated with satellite DNAs. Since begomovirus acquisition or exchange of satellite DNAs may lead to adaptation to new plant hosts and emergence of new disease complexes, it is important to investigate the diversity and distribution of these molecules. This study reports begomovirus-associated satellite DNAs identified during a vector-enabled metagenomic (VEM) survey of begomoviruses using whiteflies collected in various locations (California (USA), Guatemala, Israel, Puerto Rico, and Spain). Protein-encoding satellite DNAs, including alphasatellites and betasatellites, were identified in Israel, Puerto Rico, and Guatemala. Novel alphasatellites were detected in samples from Guatemala and Puerto Rico, resulting in the description of a phylogenetic clade (DNA-3-type alphasatellites) dominated by New World sequences. In addition, a diversity of small (~640-750 nucleotides) satellite DNAs similar to satellites associated with begomoviruses infecting Ipomoea spp. were detected in Puerto Rico and Spain. A third class of satellite molecules, named gammasatellites, is proposed to encompass the increasing number of reported small (<1 kilobase), non-coding begomovirus-associated satellite DNAs. This VEM-based survey indicates that, although recently recovered begomovirus genomes are variations of known genetic themes, satellite DNAs hold unexplored genetic diversity.