The pollen virome: A review of pollen-associated viruses and consequences for plants and their interactions with pollinators

The movement of pollen grains from anthers to stigmas, often by insect pollinator vectors, is essential for plant reproduction. However, pollen is also a unique vehicle for viral spread. Pollen-associated plant viruses reside on the outside or inside of pollen grains, infect susceptible individuals through vertical or horizontal infection pathways, and can decrease plant fitness. These viruses are transferred with pollen between plants by pollinator vectors as they forage for floral resources; thus, pollen-associated viral spread is mediated by floral and pollen grain phenotypes and pollinator traits, much like pollination. Most of what is currently known about pollen-associated viruses was discovered through infection and transmission experiments in controlled settings, usually involving one virus and one plant species of agricultural or horticultural interest. In this review, we first provide an updated, comprehensive list of the recognized pollen-associated viruses. Then, we summarize virus, plant, pollinator vector, and landscape traits that can affect pollen-associated virus transmission, infection, and distribution. Next, we highlight the consequences of plant–pollinator–virus interactions that emerge in complex communities of co-flowering plants and pollinator vectors, such as pollen-associated virus spread between plant species and viral jumps from plant to pollinator hosts. We conclude by emphasizing the need for collaborative research that bridges pollen biology, virology, and pollination biology.     

Occurrence and Diversity of Pome Fruit Viruses in Apple and Pear Orchards in Latvia

Apple chlorotic leaf spot virus (ACLSV), Apple stem pitting virus (ASPV), Apple stem grooving virus (ASGV) and Apple mosaic virus are economically important viruses infecting fruit tree species worldwide. To evaluate the occurrence of these pome fruit viruses in Latvia, a large‐scale survey was carried out in 2007. Collected samples were tested for infection by DAS ELISA and multiplex RT‐PCR. The accuracy of the detection of the viruses in multiplex RT‐PCR was confirmed by sequencing amplified PCR fragments. The results showed a wide occurrence of viruses in apple and pear commercial orchards established from non‐tested planting material. More than 89% of the tested apple trees and more than 60% of pear trees were infected with one or more pome fruit viruses. Analyses showed that the high occurrence of viruses in several apple cultivars is due to the propagation of infected clonal rootstocks and scions from infected mother trees. Sequence analyses targeting the 3′‐terminal region of the tested viruses showed various degrees of genetic diversity within respective virus isolates. This is the first report of the occurrence of ACLSV, ASGV and ASPV in apple and pear trees in Latvia and demonstrates their genetic diversity in different host genotypes.     

Xyloglucan endotransglycosylase activity during fruit development and ripening of apple and kiwifruit

Xyloglucan endotransglycosylase (XET) activity was measured in apple (Malus domestica Borkh. cv. Braeburn) pericarp and kiwifruit (Actinidia deliciosa [A. Chev.] C. F. Liang et A. R. Ferguson var. deliciosa cv. Hayward) outer pericarp and core tissues in order to establish whether a correlation exists between the activity of the enzyme and different stages of fruit development Whereas the growth rate of kiwifruit paralleled changes in XET activity throughout fruit growth, that of apple did not. Both fruits showed the highest XET activity, on a fresh weight basis, in the first two weeks after anthesis when cell division was at its highest. XET activity then decreased sharply, but as the fruit increased in size (4–8 weeks after anthesis) there was a concomitant increase in XET activity in both fruits. In the latter stage of fruit development (16–26 weeks after anthesis) XET activity increased to peak at harvest in apple fruit. During this time there was relatively little increase in fruit size and presumably therefore minimal cell expansion. XET activity then declined as fruit softened after harvest. In core tissue from kiwifruit, XET activity increased throughout the later stages of fruit growth to harvest maturity in a similar manner to apple, but continued to increase after harvest until fruit were ripe. In contrast, XET activity in the outer pericarp of kiwifruit did not increase until ripening after harvest. In apple tissue up to 30% of the XET activity was cell wall bound and could not be solubilised, even in buffer containing 2 M NaCl. The results implicate XET in cell wall assembly during cell division and expansion early in apple and kiwifruit growth. However, the disparity between apple and kiwifruit with respect to XET activity late in fruit development and ripening and the different affinities of the enzyme for the cell wall in each fruit, suggest that XET has several roles in plant development, not all of which are related to cell wall loosening during periods of accelerated growth.     

New World Bats Harbor Diverse Influenza A Viruses

Aquatic birds harbor diverse influenza A viruses and are a major viral reservoir in nature. The recent discovery of influenza viruses of a new H17N10 subtype in Central American fruit bats suggests that other New World species may similarly carry divergent influenza viruses. Using consensus degenerate RT-PCR, we identified a novel influenza A virus, designated as H18N11, in a flat-faced fruit bat (Artibeus planirostris) from Peru. Serologic studies with the recombinant H18 protein indicated that several Peruvian bat species were infected by this virus. Phylogenetic analyses demonstrate that, in some gene segments, New World bats harbor more influenza virus genetic diversity than all other mammalian and avian species combined, indicative of a long-standing host-virus association. Structural and functional analyses of the hemagglutinin and neuraminidase indicate that sialic acid is not a ligand for virus attachment nor a substrate for release, suggesting a unique mode of influenza A virus attachment and activation of membrane fusion for entry into host cells. Taken together, these findings indicate that bats constitute a potentially important and likely ancient reservoir for a diverse pool of influenza viruses.     

病毒宏基因组学研究进展

病毒宏基因组学是一种新的病毒组学研究手段,随着高通量测序技术的飞速发展,人们能够从环境中快速发现、鉴定病毒基因组的组成并研究其特征。在过去的十年里,研究者们运用病毒宏基因组学发现了许多新型病毒,增强了人们对不同环境中病毒组成、分布和多样性的了解。因此,病毒宏基因组学已成为清晰描绘各种特殊环境中病毒图谱、了解自然界中病毒分布动态的有效工具。本文主要从病毒宏基因组的概念、样品前处理和病毒总基因组提取方法、测序技术以及病毒宏基因组的应用和发展前景方面进行概述。     

Metagenomic Profiling of Viruses Associated with Rhipicephalus microplus Ticks in Yunnan Province,China

Ticks are well known as vectors of many viruses which usually do great harm to human and animal health. Yunnan Province, widely covered by flourishing vegetation and mainly relying on farming husbandry, is abundant with Rhipicephalus microplus ticks. Therefore, it is of great significance to characterize the viral profile present in R. microplus parasitizing on cattle in Yunnan Province. In this study, a total of 7387 R. microplus ticks were collected from cattle and buffalo in the northwest and southeast areas of Yunnan Province from 2015 to 2017. We investigated the virome of R.microplus using next-generation sequencing(NGS) and the prevalence of important identified viruses among tick groups by RT-PCR. It revealed the presence of diverse virus concerning chu-, rhabdo-, phlebo-, flavi-and parvo-viruses in Yunnan. These viruses consist of single-stranded, circular and segmented sense RNAs, showing a greatly diversity in genomic organization. Furthermore, continuous epidemiological survey among ticks reveals broad prevalence of three viruses(Yunnan mivirus 1, Wuhan tick vrius 1 and YN tick-associated phlebovirus 1) and two possible prevalent viruses including a flavivirus-like segmented virus(Jingmen tick virus) and a bovine hokovirus 2 in Yunnan. Serological investigation among cattle indicates that these identified viruses may be infectious to cattle and can elicit corresponding antibody. Our findings on R. microplus-associated viral community will contribute to the prevention of viral disease and tracking the viral evolution. Further analysis is needed to better elucidate the pathogenicity and natural circulation of these viruses.     

Comparison of Viromes in Ticks from Different Domestic Animals in China

Ticks are involved in the transmission of various arboviruses and some tick-borne viruses pose significant threats to the health of humans or livestock. This study aimed to investigate the geographical distribution of tick species and tickassociated viruses in central and eastern China. Total 573 ticks from domestic animals including dogs, sheep and cattle were collected in 2017. Two genera of ticks were identified including Rhipicephalus and Haemaphysalis. Sequencing was performed on Miseq Illumina platform to characterize the tick viromes from the four different sampling locations.Following trimming, 13,640 reads were obtained and annotated to 19 virus families. From these sequences, above 37.74% of the viral reads were related to the RNA viruses. Virome comparison study revealed that the tick viral diversity was considerably different in the two identified tick genera. The viral diversity of R. microplus was significantly different from that of other Rhipicephalus species. On the other hand, substantial overlap in viral species was observed between the same genera. In addition, we found no evidence that the natural host played a major role in shaping virus diversity based on the comparison of their viromes. Rather, the geographic location seems to significantly influence the viral families. Phylogenetic study indicated that the novel negative-sense RNA viruses identified in this study was closely related to Bole tick virus 1 and 3 viruses. In conclusion, the present study provides a baseline for comparing viruses detected in ticks, according to species, natural hosts, and geographic locations.     

New archaeal viruses discovered by metagenomic analysis of viral communities in enrichment cultures

Viruses infecting hyperthermophilic archaea of the phylum Crenarchaeota display enormous morphological and genetic diversity, and are classified into 12 families. Eight of these families include only one or two species, indicating sparse sampling of the crenarchaeal virus diversity. In an attempt to expand the crenarchaeal virome, we explored virus diversity in the acidic, hot spring Umi Jigoku in Beppu, Japan. Environmental samples were used to establish enrichment cultures under conditions favouring virus replication. The host diversity in the enrichment cultures was restricted to members of the order Sulfolobales. Metagenomic sequencing of the viral communities yielded seven complete or near-complete double-stranded DNA virus genomes. Six of these genomes could be attributed to polyhedral and filamentous viruses that were observed by electron microscopy in the enrichment cultures. Two icosahedral viruses represented species in the family Portogloboviridae. Among the filamentous viruses, two were identified as new species in the families Rudiviridae and Lipothrixviridae, whereas two other formed a group seemingly distinct from the known virus genera. No particle morphotype could be unequivocally assigned to the seventh viral genome, which apparently represents a new virus type. Our results suggest that filamentous viruses are globally distributed and are prevalent virus types in extreme geothermal environments.     

Incidence and detection of negative-stranded RNA viruses infecting apple and pear trees in California

Novel negative-stranded RNA (nsRNA) viruses have been recently identified in multiple agronomic crops, including pome fruit trees. Citrus concave gum-associated virus (CCGaV), citrus virus A (CiVA) and apple rubbery wood viruses 1 and 2 (ARWV1 and 2) are examples of such viruses. Given the novelty and lack of information about these pathogens in Californian orchards, in this study, real-time RT-PCR assays for CCGaV, CiVA, ARWV1 and 2 were developed and employed in a field survey. Initially, the new assays were challenged against a comprehensive set of positive and negative samples, previously analysed by high-throughput sequencing (HTS), to determine specificity. Aiming to investigate the presence of nsRNA viruses in California apple and pear orchards, 186 samples were collected from 21 different locations. As a result, 79 (42%) samples were found to be infected by these viruses in single or mixed infections. The incidence of each virus in relation to the total number of samples was 36%, 15%, 11% and 0% for ARWV2, CCGaV, ARWV1 and CiVA respectively. Overall, not considering the no detected CiVA, the other three nsRNA viruses were widely distributed among sampled orchards. To further validate the reliability of the new real-time RT-PCR assays, six samples tested positive during the survey were screened by previously described detection assays and HTS. This is the first detection of these nsRNA viruses in California, which may represent an issue in apple and pear production.     

Exploring the diversity of plant DNA viruses and their satellites using vector-enabled metagenomics on whiteflies

Current knowledge of plant virus diversity is biased towards agents of visible and economically important diseases. Less is known about viruses that have not caused major diseases in crops, or viruses from native vegetation, which are a reservoir of biodiversity that can contribute to viral emergence. Discovery of these plant viruses is hindered by the traditional approach of sampling individual symptomatic plants. Since many damaging plant viruses are transmitted by insect vectors, we have developed "vector-enabled metagenomics" (VEM) to investigate the diversity of plant viruses. VEM involves sampling of insect vectors (in this case, whiteflies) from plants, followed by purification of viral particles and metagenomic sequencing. The VEM approach exploits the natural ability of highly mobile adult whiteflies to integrate viruses from many plants over time and space, and leverages the capability of metagenomics for discovering novel viruses. This study utilized VEM to describe the DNA viral community from whiteflies (Bemisia tabaci) collected from two important agricultural regions in Florida, USA. VEM successfully characterized the active and abundant viruses that produce disease symptoms in crops, as well as the less abundant viruses infecting adjacent native vegetation. PCR assays designed from the metagenomic sequences enabled the complete sequencing of four novel begomovirus genome components, as well as the first discovery of plant virus satellites in North America. One of the novel begomoviruses was subsequently identified in symptomatic Chenopodium ambrosiodes from the same field site, validating VEM as an effective method for proactive monitoring of plant viruses without a priori knowledge of the pathogens. This study demonstrates the power of VEM for describing the circulating viral community in a given region, which will enhance our understanding of plant viral diversity, and facilitate emerging plant virus surveillance and management of viral diseases.     

贵州野生猕猴桃表型遗传多样性分析

通过对146份贵州猕猴桃种质资源表型遗传多样性的研究,为贵州猕猴桃品种选育打下基础,对146份不同区域猕猴桃种质资源48个质量性状进行描述、赋值,并结合单果重等9个数量性状,使用主成分分析、聚类分析等进行综合评价。结果表明,57个质量性状和数量性状均存在丰富的多样性和变异性;146份资源的Shannon’s遗传多样性指数为1.28,从各区域遗传多样性指数可以看出,演化进程上从遗传多样性指数高的地区向低的地区进行演化;主成分分析结果显示,前14个主成分累计贡献率达75.076%,其中前6个主成分的累计贡献率达51.911%,为提高品种选育和评价的工作效率,可将猕猴桃种质资源的描述指标简化为新梢被毛、果实被毛等18个性状;聚类结果显示,贵州猕猴桃种质的欧氏距离为1.00~25.00之间,在D=19.25处,146份野生猕猴桃分为6个类群。本文有效揭示了贵州省猕猴桃种质资源丰富的多样性和亲缘关系,明确野生猕猴桃各种群间性状区分特征和部分优良资源性状的地理分布,对贵州猕猴桃种质资源的种质鉴定、保护利用和品种选育有重要意义。     

Effects of varying pollen load on fruit set,seed set and seedling performance in apple and pear

Two apple crosses and one pear cross were carried out using three different pollen densities. In one of the apple crosses, both fruit and seed set decreased when a mixture of pollen and powder (diluted pollen treatment) was used for pollination. Also, seedlings from the undiluted treatment were more vigorous than those originating from pollination with diluted pollen. In the other apple cross, however, no effect on fruit set, seed set or growth in the first year was found. Mean diameter after the second year differed among treatments, but did not seem to be related to pollen density. For pear, the number of good seeds decreased and the number of deaf, probably unfertilized, seeds increased with decreasing pollen density. However, there was no apparent significant effect of pollen density on seedling growth. With respect to scab resistance and mildew attack in both apple crosses, no differences among treatments were found. The results indicate that gametophytic competition may occur in apple crosses, but that it is probably weaker in pear.     

中国部分地区蝙蝠携带病毒的宏基因组学分析

蝙蝠携带有60多种病毒,其中许多对人有高度致病性.为了解中国蝙蝠携带病毒的自然本底、蝙蝠病毒的多样性和挖掘潜在的病毒病原,通过基于Solexa高通量测序的病毒宏基因组学技术对从吉林、云南、湖南采集的蝙蝠组织进行病毒组学研究,获得了11 644 232条读长(Reads),并拼接出44 872条重叠序列(Contig).通过核酸序列注释发现,其中8.2％(4 002/44 872)的重叠序列与病毒相关,能进一步注释到36个病毒科,包括19种脊椎动物病毒、6种植物病毒、4种昆虫病毒和4种噬菌体.通过对重叠序列的遗传进化分析、多序列比对显示,被注释为细小病毒、腺联病毒、博卡病毒、腺病毒、小双节RNA病毒等的重叠序列与已知病毒相似,部分序列却又呈现出明显的序列差异.通过对腺病毒和博卡病毒进一步的PCR扩增证实了此研究方法可靠.旨在了解我国蝙蝠携带病毒组的构成,对建立高效的野生动物源人兽共患病的监测方法提供参考.     

The effects of pollen diversity on plant reproduction: insights from apple

For many plants, the number of pollen genotypes deposited on a flower’s stigma is positively related to fruit maturation and seed number; however, the mechanisms underlying this effect are less understood. Here we examined whether diversity of pollen (1, 3, or 5 donors) affects reproductive success in self-incompatible apple (Malus × domestica). Using paternity analysis, we then assessed the siring rate of individual donors to test whether diversity effects are related to the presence of a superior pollen donor or to donor × donor interactions. Increasing the diversity of compatible pollen enhanced seed number and reduced seed abortion in some but not all recipient genotypes. This effect was associated with an increased number of sires per fruit and non-random siring success among pollen donors. Two donors had consistently high siring rates, regardless of recipient genotype; however, the siring success of pollen donors was not correlated with their siring success in single-donor pollinations. Rather, siring success was affected by the identity of other pollen genotypes present on the stigma. Our results therefore suggest that the effects of pollen diversity are variable but may enhance fecundity by fostering interactions between pollen donors.     

Viral metagenome analysis to guide human pathogen monitoring in environmental samples

Aims: The aim of this study was to develop and demonstrate an approach for describing the diversity of human pathogenic viruses in an environmentally isolated viral metagenome. Methods and Results: In silico bioinformatic experiments were used to select an optimum annotation strategy for discovering human viruses in virome data sets and applied to annotate a class B biosolid virome. Results from the in silico study indicated that <1% errors in virus identification could be achieved when nucleotide‐based search programs (BLASTn or tBLASTx), viral genome only databases and sequence reads >200 nt were considered. Within the 51 925 annotated sequences, 94 DNA and 19 RNA sequences were identified as human viruses. Virus diversity included environmentally transmitted agents such as parechovirus, coronavirus, adenovirus and aichi virus, as well as viruses associated with chronic human infections such as human herpes and hepatitis C viruses. Conclusions: This study provided a bioinformatic approach for identifying pathogens in a virome data set and demonstrated the human virus diversity in a relevant environmental sample. Significance and Impact of the Study: As the costs of next‐generation sequencing decrease, the pathogen diversity described by virus metagenomes will provide an unbiased guide for subsequent cell culture and quantitative pathogen analyses and ensures that highly enriched and relevant pathogens are not neglected in exposure and risk assessments.     

Metagenomic Analysis of the Ferret Fecal Viral Flora

Ferrets are widely used as a small animal model for a number of viral infections, including influenza A virus and SARS coronavirus. To further analyze the microbiological status of ferrets, their fecal viral flora was studied using a metagenomics approach. Novel viruses from the families Picorna-, Papilloma-, and Anelloviridae as well as known viruses from the families Astro-, Corona-, Parvo-, and Hepeviridae were identified in different ferret cohorts. Ferret kobu- and hepatitis E virus were mainly present in human household ferrets, whereas coronaviruses were found both in household as well as farm ferrets. Our studies illuminate the viral diversity found in ferrets and provide tools to prescreen for newly identified viruses that potentially could influence disease outcome of experimental virus infections in ferrets.     

Viral transmission in honey bees and native bees,supported by a global black queen cell virus phylogeny

In recent decades, we have realized that honey bee viruses are not, in fact, exclusive to honey bees. The potential impact of Apis-affiliated viruses on native pollinators is prompting concern. Our research addresses the issue of virus crossover between honey bees and native bees foraging in the same localities. We measured the presence of black queen cell virus (BQCV), deformed wing virus (DWV) and sacbrood virus (SBV) in managed Apis mellifera (honey bees) and native Andrena spp. (subgenus Melandrena) bee populations in five commercial orchards. We identified viral presence across sites and bees and related these data to measures of bee community diversity. All viruses were found in both managed and native bees, and BQCV was the most common virus in each. To establish evidence for viral crossover between taxa, we undertook an additional examination of BQCV where 74 samples were sequenced and placed in a global phylogenic framework of hundreds of BQCV strains. We demonstrate pathogen sharing across managed honey bees and distantly related wild bees. This phylogenetic analysis contributes to growing evidence for host switching and places local incidence patterns in a worldwide context, revealing multispecies viral transmission.