Almond Can Be Infected by Plum Pox Virus-D Isolate Penn4 and Is a Transmission-Competent Host.

Although it is currently eradicated from the United States, Plum pox virus (PPV) poses an ongoing threat to U.S. stone fruit production. Although almond (Prunus dulcis) is known to be largely resistant to PPV, there is conflicting evidence about its potential to serve as an asymptomatic reservoir host for the virus and thus serve as a potential route of entry. Here, we demonstrate that both Tuono and Texas Mission cultivars can be infected by the U.S. isolate PPV Dideron (D) Penn4 and that Tuono is a transmission-competent host, capable of serving as a source of inoculum for aphid transmission of the virus. These findings have important implications for efforts to keep PPV out of the United States and highlight the need for additional research to test the susceptibility of almond to other PPV-D isolates.

Viral Reservoir Capacity of Wild Prunus Alternative Hosts of Plum Pox Virus Through Multiple Cycles of Transmission and Dormancy.

Plum pox virus (PPV) is a significant pathogen of Prunus worldwide and is known for having a broad experimental host range. Many of these hosts represent epidemiological risks as potential wild viral reservoirs. A comparative study of the PPV reservoir capacity of three commonly found native North American species, western choke cherry (Prunus virginiana var. demissa), black cherry (Prunus serotina), and American plum (Prunus americana) was conducted. Pennsylvania isolates of PPV-D were transmitted from the original host peach (Prunus persica cv. GF305) to all three species. Viral accumulation and transmission rates to alternative hosts and peach were monitored over the course of five vegetative growth and cold induced dormancy (CID) cycles. The three alternative host species demonstrated differences in their ability to maintain PPV-D and the likelihood of transmission to additional alternative hosts or back transmission to peach. Western choke cherry had low (5.8%) initial infection levels, PPV-D was not transmissible to additional western choke cherry, and transmission of PPV-D from western choke cherry to peach was only possible before the first CID cycle. Black cherry had intermediate initial infection levels (26.6%) but did not maintain high infection levels after repeated CID cycles. Conversely, American plum had a high level (50%) of initial infection that was not significantly different from initial infection in peach (72.2%) and maintained moderate levels (15 to 25%) of infection and PPV-D transmission to both American plum and peach through all five cycles of CID. Our results indicate that American plum has the greatest potential to act as a reservoir host for Pennsylvania isolates of PPV-D.

Dynamic changes impact the plum pox virus population structure during leaf and bud development.

Plum pox virus (PPV) is a worldwide threat to stone fruit production. Its woody perennial hosts provide a dynamic environment for virus evolution over multiple growing seasons. To investigate the impact seasonal host development plays in PPV population structure, next generation sequencing of ribosome associated viral genomes, termed translatome, was used to assess PPV variants derived from phloem or whole leaf tissues over a range of plum leaf and bud developmental stages. Results show that translatome PPV variants occur at proportionately higher levels in bud and newly developing leaf tissues that have low infection levels while more mature tissues with high infection levels display proportionately lower numbers of viral variants. Additional variant analysis identified distinct groups based on population frequency as well as sets of phloem and whole tissue specific variants. Combined, these results indicate PPV population dynamics are impacted by the tissue type and developmental stage of their host.

Translatome Profiling of Plum Pox Virus-Infected Leaves in European Plum Reveals Temporal and Spatial Coordination of Defense Responses in Phloem Tissues.

Plum pox virus (PPV) is the causative agent of sharka, a devastating disease of stone fruits including peaches, apricots, and plums. PPV infection levels and associated disease symptoms can vary greatly, depending upon the virus strain, host species, or cultivar as well as developmental age of the infected tissues. For example, peaches often exhibit mild symptoms in leaves and fruit while European plums typically display severe chlorotic rings. Systemic virus spread into all host tissues occurs via the phloem, a process that is poorly understood in perennial plant species that undergo a period of dormancy and must annually renew phloem tissues. Currently, little is known about how phloem tissues respond to virus infection. Here, we used translating ribosome affinity purification followed by RNA sequencing to identify phloem- and nonphloem-specific gene responses to PPV infection during leaf development in European plum (Prunus domestica L.). Results showed that, during secondary leaf morphogenesis (4- and 6-week-old leaves), the phloem had a disproportionate response to PPV infection with two- to sixfold more differentially expressed genes (DEGs) in phloem than nonphloem tissues, despite similar levels of viral transcripts. In contrast, in mature 12-week-old leaves, virus transcript levels dropped significantly in phloem tissues but not in nonphloem tissues. This drop in virus transcripts correlated with an 18-fold drop in phloem-specific DEGs. Furthermore, genes associated with defense responses including RNA silencing were spatially coordinated in response to PPV accumulation and were specifically induced in phloem tissues at 4 to 6 weeks. Combined, these findings highlight the temporal and spatial dynamics of leaf tissue responses to virus infection and reveal the importance of phloem responses within a perennial host.

Nanopore Sequencing as a Surveillance Tool for Plant Pathogens in Plant and Insect Tissues.

Plant pathogens are constantly emerging and spreading into new areas and there are often limited postdiagnosis treatment options for infection, making surveillance key to their control. Here we present results from a study testing the efficacy of a portable nanopore-based massively parallel sequencing (MPS) technology for use in the detection of diverse plant pathogens in selected samples. The Oxford MinION device was coupled with whole transcriptome amplification (WTA) to sequence the metatranscriptome of plant and insect tissues infected with either Candidatus Liberibacter asiaticus or plum pox virus. Results showed that this methodology is useful for detecting unsuspected viral and bacterial pathogens in plant and insect tissues. The percentage of generated reads assigned to plum pox virus was 95% from infected tissue and 3% from the viruliferous insect, Myzus persicae. Diaphorina citri sequencing led to 22% of the reads mapping as Ca. L. asiaticus. Plum pox virus and Ca. L. asiaticus were detected in both tissue and insect samples near the beginning of each sequencing run, demonstrating the capability of this methodology to obtain results rapidly. This approach also proved the capability of this system to determine the major components of the insect vector's microbiome and the specific strain of small-genome, high-titer pathogens.

Host Adaptation of Soybean Dwarf Virus Following Serial Passages on Pea (Pisum sativum) and Soybean (Glycine max).

Soybean Dwarf Virus (SbDV) is an important plant pathogen, causing economic losses in soybean. In North America, indigenous strains of SbDV mainly infect clover, with occasional outbreaks in soybean. To evaluate the risk of a US clover strain of SbDV adapting to other plant hosts, the clover isolate SbDV-MD6 was serially transmitted to pea and soybean by aphid vectors. Sequence analysis of SbDV-MD6 from pea and soybean passages identified 11 non-synonymous mutations in soybean, and six mutations in pea. Increasing virus titers with each sequential transmission indicated that SbDV-MD6 was able to adapt to the plant host. However, aphid transmission efficiency on soybean decreased until the virus was no longer transmissible. Our results clearly demonstrated that the clover strain of SbDV-MD6 is able to adapt to soybean crops. However, mutations that improve replication and/or movement may have trade-off effects resulting in decreased vector transmission.

A previously undescribed potyvirus isolated and characterized from arborescent Brugmansia.

A suspected virus disease was identified from an arborescent Brugmansia x candida Pers. (syn. Datura candida Pers.) tree. The causal agent was aphid transmissible at low rates. Viral particles were purified from infected tobacco tissue, analyzed, and purified virions were inoculated into healthy tobacco plants to recreate the symptoms. The virions had a mean length of 720-729 nm, and infected cells contained inclusion bodies typical of potyvirus infections. Analysis of infected tissues and purified virions with a panel of potyvirus-specific antibodies confirmed identification as a potyvirus. Viral host range, dilution end point, thermal tolerance and aphid transmission characteristics were examined. The viral genome (9761 nt) is typical of potyviruses, with the closest related potyvirus being pepper mottle virus, at 72 % nt sequence identity. Based on conventions for naming novel potyviruses, the virus was determined to be a member of a previously undescribed species, tentatively named "Brugmansia mosaic virus" (BruMV).

Molecular, ultrastructural, and biological characterization of Pennsylvania isolates of Plum pox virus.

Plum pox virus (PPV) was identified in Pennsylvania in 1999. The outbreak was limited to a four-county region in southern Pennsylvania. Initial serological and molecular characterization indicated that the isolates in Pennsylvania belong to the D strain of PPV. The Pennsylvania isolates were characterized by sequence analysis, electron microscopy, host range, and vector transmission to determine how these isolates related to their previously studied European counterparts. Genetically, Pennsylvania (PPV-Penn) isolates were more closely related to each other than to any other PPV-D strains, and isolates from the United States, Canada, and Chile were more closely related to each other than to European isolates. The PPV-Penn isolates exist as two clades, suggesting the possibility of multiple introductions. Electron microscopy analysis of PPV-Penn isolates, including cytopathological studies, indicated that the virions were similar to other Potyvirus spp. PPV-Penn isolates had a herbaceous host range similar to that of European D isolates. There were distinct differences in the transmission efficiencies of the two PPV-Penn isolates using Myzus persicae and Aphis spiraecola as vectors; however, both PPV-Penn isolates were transmitted by M. persicae more efficiently than a European D isolate but less efficiently than a European M isolate.

Molecular analysis of soybean dwarf virus isolates in the eastern United States confirms the presence of both D and Y strains and provides evidence of mixed infections and recombination.

Soybean dwarf virus (SbDV), first identified as an agricultural problem in Japan, has emerged as a growing problem in the Midwestern United States. The majority of research on SbDV had been limited to four lab maintained strains from Japan. SbDV had been found in clover in the eastern United States, but these isolates rarely emerged into soybeans. These isolates were analyzed by multiplex PCR and sequencing, revealing that some were infections of both Y and D components, including a recombinant subisolate. Phylogenetic analyses for the US isolates revealed a broad diversity of SbDV, with selection pressure greater on the movement protein than the coat protein. The field isolates from the Eastern United States showed differences in symptoms, aphid transmission and host range, demonstrating that a study of field isolates is an important complement to laboratory maintained strains in understanding the biology and evolution of plant viruses.

Real-time identification of serious infection in geriatric patients using clinical information system surveillance.

OBJECTIVES: To develop and characterize an automated syndromic surveillance mechanism for early identification of older emergency department (ED) patients with possible life-threatening infection. DESIGN: Prospective, consecutive-enrollment, single-site observational study. SETTING: A large university medical center with an annual ED census of 75,273. PARTICIPANTS: Patients aged 70 and older admitted to the ED and having two or more systemic inflammatory response syndrome (SIRS) criteria during their ED stay. MEASUREMENTS: A search algorithm was developed to screen the census of the ED through its clinical information system. A study coordinator confirmed all patients electronically identified as having a probable infectious explanation for their visit. RESULTS: Infection accounted for 28% of ED and 34% of final hospital diagnoses. Identification using the software tool alone carried a 1.63 relative risk of infection (95% confidence interval CI51.09-2.44) compared with other ED patients sufficiently ill to require admission. Follow-up confirmation by a study coordinator increased the risk to 3.06 (95% CI52.11-4.44). The sensitivity of the strategy overall wasmodest (14%), but patients identified were likely to have an infectious diagnosis (specificity 598%). The most common SIRS criterion triggering the electronic notification was the combination of tachycardia and tachypnea. CONCLUSION: A simple clinical informatics algorithm can detect infection in elderly patients in real time with high specificity. The utility of this tool for research and clinical care may be substantial.

Adaptation of plum pox virus to a herbaceous host (Pisum sativum) following serial passages.

Plum pox virus (PPV) populations from peaches are able to adapt consistently to herbaceous hosts, characterized by a reduction in time to symptom development, increases in inoculation efficiency and increased titres. PPV adaptation was studied by using pea (Pisum sativum) as an alternative host. Two isolates of PPV from peaches were inoculated and passaged in peas ten times using either aphid or mechanical inoculation, generating four independent passage lines. Mechanical-transmission efficiency from peach to pea improved from 3 % at passage 1 to 100 % by serial passage 4 on peas. Inoculation using aphid vectors required six to ten serial passages in pea to reach a peak of 50-60 % transmission efficiency. Sequence analyses of all four PPV population lines inoculated sequentially to pea identified a specific mutation occurring consistently in the NIb gene when compared with the same PPV isolates passaged in parallel in peach. The mutation allowed PPV to replicate up to 20 times faster in the new host. Pea-adapted strains of PPV at every passage were also tested for their ability to infect the original host, peach. Regardless of the number of previous passages, all pea-adapted PPV strains consistently infected peach at low levels using aphid inoculation.

Analysis of genetic bottlenecks during horizontal transmission of Cucumber mosaic virus.

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.

Specific detection and quantification of Plum pox virus by real-time fluorescent reverse transcription-PCR.

Plum pox virus (PPV), a destructive and economically devastating pathogen of Prunus species, was recently discovered in Pennsylvania and Canada. Current containment efforts involve eradication of infected trees based on ELISA surveys, which are laborious and less sensitive than PCR-based techniques. A real-time, fluorescent, reverse transcription-polymerase chain reaction (RT-PCR) assay was developed for the detection of PPV in the Smart Cycler (Cepheid). The methods developed are reproducible, specific to PPV, and sensitive enough to consistently detect PPV transcripts at the 10-20 fg level. The assay is more sensitive than either ELISA or traditional PCR followed by visualization with ethidium-bromide. PPV was detected from multiple hosts and from multiple Prunus tissues (leaf, stem, bud, and root). A dilution series using an in vitro synthesized transcript containing the target sequence as a standard demonstrated that the assay was effective for quantitation of viral template. The real-time PCR assay is a valuable tool for PPV detection and liter quantification in field or laboratory settings.