William L. Schneider

Genetic Diversity in RNA Virus Quasispecies Is Controlled by Host-Virus Interactions

ABSTRACT Many RNA viruses have genetically diverse populations known as quasispecies. Important biological characteristics may be related to the levels of diversity in the quasispecies (quasispecies cloud size), including adaptability and host range. Previous work usingTobacco mosaic virus and Cucumber mosaic virusindicated that evolutionarily related viruses have very different levels of diversity in a common host. The quasispecies cloud size for these viruses remained constant throughout serial passages. Inoculation of these viruses on a number of hosts demonstrated that quasispecies cloud size is not constant for these viruses but appears to be dependent on the host. The quasispecies cloud size remained constant as long as the viruses were maintained on a given host. Shifting the virus between hosts resulted in a change in cloud size to levels associated with the new host. Quasispecies cloud size for these viruses is related to host-virus interactions, and understanding these interactions may facilitate the prediction and prevention of emerging viral diseases.

Analysis of Genetic Bottlenecks during Horizontal Transmission of Cucumber Mosaic Virus

ABSTRACT Genetic bottlenecks may occur in virus populations when only a few individuals are transferred horizontally from one host to another, or when a viral population moves systemically from the infection site. Genetic bottlenecks during the systemic movement of an RNA plant virus population were reported previously (H. Li and M. J. Roossinck, J. Virol. 78:10582-10587, 2004). In this study we mechanically inoculated an artificial population consisting of 12 restriction enzyme marker mutants of Cucumber mosaic virus (CMV) onto young leaves of squash plants and used two aphid species, Aphis gossypii and Myzus persicae, to transmit the virus populations from infected source plants to healthy squash plants. Horizontal transmission by aphids constituted a significant bottleneck, as the population in the aphid-inoculated plants contained far fewer mutants than the original inoculum source. Additional experiments demonstrated that genetic variation in the artificial population of CMV is not reduced during the acquisition of the virus but is significantly reduced during the inoculation period.

Evolutionarily Related Sindbis-Like Plant Viruses Maintain Different Levels of Population Diversity in a Common Host

ABSTRACT The levels of population diversity of three related Sindbis-like plant viruses, Tobacco mosaic virus (TMV), Cucumber mosaic virus (CMV), and Cowpea chlorotic mottle virus(CCMV), in infections of a common host, Nicotiana benthamiana, established from genetically identical viral RNA were examined. Despite probably having a common evolutionary ancestor, the three viruses maintained different levels of population diversity. CMV had the highest levels of diversity, TMV had an intermediate level of diversity, and CCMV had no measurable level of diversity in N. benthamiana. Interestingly, the levels of diversity were correlated to the relative host range sizes of the three viruses. The levels of diversity also remained relatively constant over the course of serial passage. Closer examination of the CMV and TMV populations revealed biases for particular types of substitutions and regions of the genome that may tolerate fewer mutations.

Environment Determines Fidelity for an RNA Virus Replicase

ABSTRACT The rate of insertion and deletion mutations of the replicase of Cucumber mosaic virus (CMV) was determined in planta by using a parasitic satellite RNA (satRNA) as a reporter. We found that the CMV replicase had different fidelity in different environments, with important implications in viral disease evolution. Insertions were very rare events, irrespective of the region of the satRNA genome assayed and independent of the hosts tested. On the other hand, deletion events were more frequent but were restricted to a highly structured region of the reporter. Deletion mutation rates were different for the two hosts tested, although the mutation distribution was not influenced by the hosts. Moreover, hot spots with high mutation rates were identified on the satRNA genome.

Plum Pox in North America: Identification of Aphid Vectors and a Potential Role for Fruit in Virus Spread

ABSTRACT Thirteen aphid species were tested for their ability to transmit Pennsylvania isolates of Plum pox virus (PPV) collected in Columbia (PENN-3), Franklin (PENN-4), and York (PENN-7) Counties, PA. Four species, Aphis fabae, A. spiraecola, Brachycaudus persicae, and Myzus persicae, consistently transmitted PPV in preliminary transmission tests. Two species, Metopolophium dirhodum and Rhopalosiphum padi, were occasional inefficient vectors. Toxoptera citricida, from Florida, also was an effective vector but it does not occur in major stone-fruit-growing states. Species not transmitting PPV in parallel tests included Acyrthosiphon pisum, Aphis glycines, Aulacorthum solani, Macrosiphum euphorbiae, Rhopalosiphum maidis, and Sitobion avenae. When given a 3-day probing access period simultaneously on PPV-infected peach seedlings and healthy peach seedlings, Myzus persicae, Aphis spiraecola, A. fabae, and B. persicae transmitted PPV to 63, 31, 38, and 32% of the healthy peach seedlings, respectively. When given a similar probing period on PPV-infected peach fruit and healthy peach seedlings, the same aphid species transmitted PPV to 50, 35, 0, and 0% of seedlings, respectively. Results support the hypothesis of secondary PPV spread by indigenous aphids in Pennsylvania, and suggest that PPV-infected fruit has the potential to function as a virus source for long-distance dispersal.

Mutant clouds and occupation of sequence space in plant RNA viruses.

In nature, RNA viruses of plants often must adapt to ever-changing environments in the form of frequent host switches. This would favor a highly diverse population for transmission. However, most viruses that have been studied have been viruses of monocultural crops. In crop viruses, the mutation frequency of individual viral quasispecies varies greatly, both in experiment evolution studies and in populations of viruses within single field plants. There is some correlation between host range and mutation frequency in experimental evolution studies, but few viruses have been examined at the individual quasispecies level. Many questions about the nature of plant RNA virus populations and factors that affect the effective population sizes, such as genetic bottlenecks and postive and negative selection, have only begun to be studied. Many more analyses are required before generalized patterns can be determined.

Role Bending: Complex Relationships Between Viruses, Hosts, and Vectors Related to Citrus Leprosis, an Emerging Disease

Citrus leprosis complex is an emerging disease in the Americas, associated with two unrelated taxa of viruses distributed in South, Central, and North America. The cytoplasmic viruses are Citrus leprosis virus C (CiLV-C), Citrus leprosis virus C2 (CiLV-C2), and Hibiscus green spot virus 2, and the nuclear viruses are Citrus leprosis virus N (CiLV-N) and Citrus necrotic spot virus. These viruses cause local lesion infections in all known hosts, with no natural systemic host identified to date. All leprosis viruses were believed to be transmitted by one species of mite, Brevipalpus phoenicis. However, mites collected from CiLV-C and CiLV-N infected citrus groves in Mexico were identified as B. yothersi and B. californicus sensu lato, respectively, and only B. yothersi was detected from CiLV-C2 and CiLV-N mixed infections in the Orinoco regions of Colombia. Phylogenetic analysis of the helicase, RNA-dependent RNA polymerase 2 domains and p24 gene amino acid sequences of cytoplasmic leprosis viruses showed a close relationship with recently deposited mosquito-borne negevirus sequences. Here, we present evidence that both cytoplasmic and nuclear viruses seem to replicate in viruliferous Brevipalpus species. The possible replication in the mite vector and the close relationship with mosquito borne negeviruses are consistent with the concept that members of the genus Cilevirus and Higrevirus originated in mites and citrus may play the role of mite virus vector.

E-probe Diagnostic Nucleic acid Analysis (EDNA): A theoretical approach for handling of next generation sequencing data for diagnostics

Plant biosecurity requires rapid identification of pathogenic organisms. While there are many pathogen-specific diagnostic assays, the ability to test for large numbers of pathogens simultaneously is lacking. Next generation sequencing (NGS) allows one to detect all organisms within a given sample, but has computational limitations during assembly and similarity searching of sequence data which extend the time needed to make a diagnostic decision. To minimize the amount of bioinformatic processing time needed, unique pathogen-specific sequences (termed e-probes) were designed to be used in searches of unassembled, non-quality checked, sequence data. E-probes have been designed and tested for several selected phytopathogens, including an RNA virus, a DNA virus, bacteria, fungi, and an oomycete, illustrating the ability to detect several diverse plant pathogens. E-probes of 80 or more nucleotides in length provided satisfactory levels of precision (75%). The number of e-probes designed for each pathogen varied with the genome size of the pathogen. To give confidence to diagnostic calls, a statistical method of determining the presence of a given pathogen was developed, in which target e-probe signals (detection signal) are compared to signals generated by a decoy set of e-probes (background signal). The E-probe Diagnostic Nucleic acid Analysis (EDNA) process provides the framework for a new sequence-based detection system that eliminates the need for assembly of NGS data.

Genome Assembly of Citrus Leprosis Virus Nuclear Type Reveals a Close Association with Orchid Fleck Virus

ABSTRACT The complete genome of citrus leprosis virus nuclear type (CiLV-N) was identified by small RNA sequencing utilizing leprosis-affected citrus samples collected from the state of Querétaro, Mexico. The nucleotide identity and phylogenetic analysis indicate that CiLV-N is very closely related to orchid fleck virus, which typically infects Cymbidium species.

A Case Study on Discovery of Novel Citrus Leprosis Virus Cytoplasmic Type 2 Utilizing Small RNA Libraries by Next Generation Sequencing and Bioinformatic Analyses

The advent of innovative sequencing technology referred to as “Next-Generation” Sequencing (NGS), provides a new approach to identify the ‘unknown known’ and ‘unknown unknown’ viral pathogens without a priori knowledge. The genomes of plant viruses can be rapidly determined even when occurring at extremely low titers in the infected host. The method is based on massively parallel sequencing of the population of small RNA molecules 18-35 nucleotides in length produced by RNA silencing host defense. Improvements in chemistries, bioinformatic tools and advances in engineering has reduced the costs of NGS, increased its accessibility, and enabled its application in the field of plant virology. In this review, we discuss the utilization of the Illumina GA IIX platform combined with the application of molecular biology and bioinformatic tools for the discovery of a novel cytoplasmic Citrus leprosis virus (CiLV). This new virus produced symptoms typical of CiLV but was not detected with either serological or PCR-based assays for the previously described virus. The new viral genome was also present in low titer in sweet orange (Citrus sinensis), an important horticultural crop with incomplete genomic resources. This is a common situation in horticultural research and provides an example of the broader utility of this approach. In addition to the discovery of novel viruses, the sequence data may be useful for studies of viral evolution and ecology and the interactions between viral and host transcriptomes.