The transmission mode of grapevine red blotch virus (GRBV, genus Grablovirus, family Geminiviridae) by Spissistilus festinus, the three-cornered alfalfa hopper, is unknown. By analogy with other members in the family Geminiviridae, we hypothesized circulative, nonpropagative transmission. Time course experiments revealed GRBV in dissected guts, hemolymph and heads with salivary glands following a 5-, 8- and 10-day exposure to infected grapevines, respectively. After a 15-day acquisition on infected grapevines and subsequent transfer on alfalfa, a non-host of GRBV, the virus titer decreased over time in adult insects, as shown by qPCR. Snap bean proved to be a feeding host of S. festinus and a pseudo-systemic host of GRBV following Agrobacterium tumefaciens-mediated delivery of an infectious clone. The virus was efficiently transmitted by S. festinus from infected snap bean plants to excised snap bean trifoliates (90%) or grapevine leaves (100%) but less efficiently from infected grapevine plants to excised grapevine leaves (10%) or snap bean trifoliates (67%). Transmission of GRBV also occurred transstadially but not via seeds. The virus titer was significantly higher in guts and hemolymph relative to heads with salivary glands, and in adults emanating from third compared with first instars that emerged on infected grapevine plants and developed on snap bean trifoliates. This study demonstrated circulative, nonpropagative transmission of GRBV by S. festinus with an extended acquisition access period compared with other viruses in the family Geminiviridae and marked differences in transmission efficiency between grapevine, the natural host, and snap bean, an alternative herbaceous host.

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Glutathione is an important biological molecule which can be an indicator of numerous diseases. A method for self-powered detection of glutathione levels in solution has been developed using an enzymatic biofuel cell. The device consists of a glucose oxidase anode and a bilirubin oxidase cathode. For the detection of glutathione, the inhibition of bilirubin oxidase leads to a measurable decrease in current and power output. The reported method has a detection limit of 0.043 mM and a linear range up to 1.7 mM. Being able to detect a range of concentrations can be useful in evaluating a patient’s health. This method has the potential to be implemented as a quick, low-cost alternative to previously reported methods.

Viruses can elicit varying types and severities of symptoms during plant host infection. We investigated changes in the proteome and transcriptome of Nicotiana benthamiana plants infected by grapevine fanleaf virus (GFLV) with an emphasis on vein clearing symptom development. Comparative, time-course liquid chromatography tandem mass spectrometry and 3′ ribonucleic acid sequencing analyses of plants infected by two wildtype GFLV strains, one symptomatic and one asymptomatic, and their asymptomatic mutant strains carrying a single amino acid change in the RNA-dependent RNA polymerase (RdRP) were conducted to identify host biochemical pathways involved in viral symptom development. During peak vein clearing symptom display at 7 days post-inoculation (dpi), protein and gene ontologies related to immune response, gene regulation, and secondary metabolite production were overrepresented when contrasting wildtype GFLV strain GHu and mutant GHu-1EK802G Pol. Prior to the onset of symptom development at 4 dpi and when symptoms faded away at 12 dpi, protein and gene ontologies related to chitinase activity, hypersensitive response, and transcriptional regulation were identified. This systems biology approach highlighted how a single amino acid of a plant viral RdRP mediates changes to the host proteome (∼1%) and transcriptome (∼8.5%) related to transient vein clearing symptoms and the network of pathways involved in the virus–host arms race.

Deer tick virus (DTV) is an emerging pathogen in North America. This virus can cause nervous system complications such as encephalitis in humans. Further, no data has been surmounted around long-term effects of infection from DTV patients across variable age groups. Diagnostic tools of DTV used by government laboratories are based on RT-PCR using patient serum or ticks. This paper explores the feasibility of a colorimetric loop-mediated isothermal amplification (LAMP) assay to create a point-of-care diagnostic methodology for use in field and in primary care. LAMP consists of six primers that bind to target DNA and amplifies variable length nucleotide strands that can be visualized through side reactions or via electrophoresis. First, a viable LAMP primer set, and a primer set that dimerizes and amplifies DNA regardless of compatibility were created in silico and validated in vitro. Then, a specific LAMP assay was developed. Our findings showed this method can be performed within 30 minutes and can measure with limits of detection comparable to PCR.

Nicotiana benthamiana is extensively used as a model herbaceous plant for virus-host interactions and sytems biology research. Here we describe a methodology to phenotype the root system architecture of N. benthamiana plants following infection with grapevine fanleaf virus, an economically important soil borne virus that is present in most vineyards worldwide. Upon completion of this protocol, images of whole root crowns can be analyzed with root phenotyping software. Following image acquisition, root tissue can be further processed for cross sectioning and ribonucleic acid extraction. Additional data (plant height, dry biomass, and leaf symptoms) were collected for correlation analysis. Documentation of root system architecture is an important first step to understand how a virus can manipulate its plant host below ground when most research is focusing on disease symptomology in leaves and fruits. This protocol allows for utilization of other viruses that infect N. benthamiana and adaptation to other plant hosts.

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The amplification of nucleic acids is a fundamental tool utilized in various scientific disciplines, including Molecular Biology, Immunology, Microbiology, and Genetics. However, due to the time and technology required for traditional polymerase chain reaction and its derivatives, it is not always possible to include such methodologies in undergraduate laboratory curricula. Loopmediated isothermal amplification (LAMP), a technology that has become increasingly utilized in a variety of laboratory and field settings during the past two decades, is an alternate method of nucleic acid amplification that is rapid, sensitive, and performed under isothermal conditions. We describe an adaptable, inquiry-driven laboratory module that is focused on the detection of Escherichia coli DNA via LAMP amplification. The main objectives of the module are to introduce students to the principles and protocols of LAMP, to help students develop the ability to apply the scientific method to scientific questions, to guide students as they develop the ability to identify the most appropriate methodology to use in the investigation of scientific questions, and to train students to critically evaluate scientific data, theories, and principles and to articulate their evaluations in both written and oral formats.