Genome Equivalent Copies Research Articles

Landscape structure affects temporal dynamics in the bumble bee virome: Landscape heterogeneity supports colony resilience

Virus spillovers from managed honey bees, Apis mellifera, are thought to contribute to the decline of wild pollinators, including bumble bees. However, data on the impact of such viruses on wild pollinators remain scarce, and the influence of landscape structure on virus dynamics is poorly understood. In this study, we deployed bumble bee colonies in an agricultural landscape and studied changes in the bumble bee virome during field placement under varying habitat composition and configuration using a multiscale analytical framework. We estimated prevalence of viruses and viral loads (i.e. number of viral genomic equivalent copies) in bumble bees before and after placing them in the field using next generation sequencing and quantitative PCR. The results show that viral loads and number of different viruses present increased during placement in the field and that the virus composition of the colonies shifted from an initial dominance of honey bee associated viruses to a higher number (in both viral loads and number of viruses present) of bumble bee associated viruses. Especially DWV-B, typical for honey bees, drastically decreased after the time in the field. Viral loads prior to placing colonies in the field showed no effect on colony development, suggesting low impacts of these viruses in field settings. Notably, we further demonstrate that increased habitat diversity results in a lower number of different viruses present in Bombus colonies, while colonies in areas with well-connected farmland patches decreased in their total viral load after field placement. Our results emphasize the importance of landscape heterogeneity and connectivity for wild pollinator health and that these influences predominate at fine spatial scales.

Development of a novel sequence based real-time PCR assay for specific and sensitive detection of Burkholderia pseudomallei in clinical and environmental matrices

BackgroundMelioidosis, caused by the category B biothreat agent Burkholderia pseudomallei, is a disease with a high mortality rate and requires an immediate culture-independent diagnosis for effective disease management. In this study, we developed a highly sensitive qPCR assay for specific detection of Burkholderia pseudomallei and melioidosis disease diagnosis based on a novel target sequence.MethodsAn extensive in-silico analysis was done to identify a novel and highly conserved sequence for developing a qPCR assay. The specificity of the developed assay was analyzed with 65 different bacterial cultures, and the analytical sensitivity of the assay was determined with the purified genomic DNA of B. pseudomallei. The applicability of the assay for B. pseudomallei detection in clinical and environmental matrices was evaluated by spiking B. pseudomallei cells in the blood, urine, soil, and water along with suitable internal controls.ResultsA novel 85-nucleotide-long sequence was identified using in-silico tools and employed for the development of the highly sensitive and specific quantitative real-time PCR assay S664. The assay S664 was found to be highly specific when evaluated with 65 different bacterial cultures related and non-related to B. pseudomallei. The assay was found to be highly sensitive, with a detection limit of 3 B. pseudomallei genome equivalent copies per qPCR reaction. The detection limit in clinical matrices was found to be 5 × 102 CFU/mL for both human blood and urine. In environmental matrices, the detection limit was found to be 5 × 101 CFU/mL of river water and 2 × 103 CFU/gm of paddy field soil.ConclusionsThe findings of the present study suggest that the developed assay S664 along with suitable internal controls has a huge diagnostic potential and can be successfully employed for specific, sensitive, and rapid molecular detection of B. pseudomallei in various clinical and environmental matrices.

Evaluation of a novel chlorine dioxide-based packaging technology to reduce human enteric virus contamination on refrigerated tomatoes and blueberries

IntroductionChlorine dioxide (ClO2) is a promising antimicrobial with various food applications, one of those being inclusion in packaging. The purpose of this study was to evaluate a novel ClO2-based antimicrobial packaging system (InvisiShield™) for its efficacy against human norovirus (hNoV) and hepatitis A virus (HAV) in refrigerated fresh produce.MethodsGrape tomatoes or blueberries were placed in polypropylene trays and selectively inoculated with 6.0 log10 hNoV Genome Equivalent Copies (GEC; 20% stool suspension) or 6.2 log10 HAV GEC (cell culture lysate). Trays were heat sealed with a three-phase polymer film consisting of a base, channeling agent, and the ClO2 active (treatment); or control (no active) film and stored at 7°C for 24, 48 h, and 7 days. At each timepoint, the product was collected and processed for virus concentration using the sequential steps of elution and polyethylene glycol precipitation. Viruses in extracts were quantified using RNase-RT-qPCR.Results and discussionLog10 reductions (LR) in hNoV GEC for tomatoes were 2.2 ± 1.3, 2.9 ± 0.7, and 3.6 ± 0.3, after 24, 48 h and 7 days, respectively. For blueberries, hNoV LR were 1.4 ± 0.7, 1.7 ± 0.5, and 2.7 ± 0.2 GEC, respectively. Hepatitis A virus GEC LR were 0.4 ± 0.2, 1.0 ± 0.1, and 2.1 ± 0.7 for tomatoes, and 0.1 ± 0.2, 1.2 ± 0.4, and 3.2 ± 0.2 for blueberries, after 24, 48 h and 7 days, respectively. Position of the fruit in the tray did not affect inactivation (p > 0.05). Sensory analysis on the treated tomato products revealed no statistically significant difference in appearance, flavor and texture attributes compared to the control. This novel ClO2-based antimicrobial packaging system effectively reduced concentrations of hNoV and HAV, as evaluated using reduction in GEC as proxy for infectivity, on grape tomatoes and blueberries after one day, with efficacy improving over 7 days of refrigerated storage. This technology shows promise as an antiviral treatment as applied to refrigerated fresh produce items.

Effect of Norovirus Inoculum Dose on Virus Kinetics, Shedding, and Symptoms.

The effect of norovirus dose on outcomes such as virus shedding and symptoms after initial infection is not well understood. We performed a secondary analysis of a human challenge study by using Bayesian mixed-effects models. As the dose increased from 4.8 to 4,800 reverse transcription PCR units, the total amount of shed virus in feces increased from 4.5 × 1011 to 3.4 × 1012 genomic equivalent copies; in vomit, virus increased from 6.4 × 105 to 3.0 × 107 genomic equivalent copies. Onset time of viral shedding in feces decreased from 1.4 to 0.8 days, and time of peak viral shedding decreased from 2.3 to 1.5 days. Time to symptom onset decreased from 1.5 to 0.8 days. One type of symptom score increased. An increase in norovirus dose was associated with more rapid shedding and symptom onset and possibly increased severity. However, the effect on virus load and shedding was inconclusive.

Dependency of sanitation infrastructure on the discharge of faecal coliform and SARS-CoV-2 viral RNA in wastewater from COVID and non-COVID hospitals in Dhaka, Bangladesh

The detection of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) RNA in wastewater can be used as an indicator of the presence of SARS-CoV-2 infection in specific catchment areas. We conducted a hospital-based study to explore wastewater management in healthcare facilities and analyzed SARS-CoV-2 RNA in the hospital wastewater in Dhaka city during the Coronavirus disease (COVID-19) outbreak between September 2020–January 2021. We selected three COVID-hospitals, two non-COVID-hospitals, and one non-COVID-hospital with COVID wards, conducted spot-checks of the sanitation systems (i.e., toilets, drainage, and septic-tank), and collected 90 untreated wastewater effluent samples (68 from COVID and 22 from non-COVID hospitals). E. coli was detected using a membrane filtration technique and reported as colony forming unit (CFU). SARS-CoV-2 RNA was detected using the iTaq Universal Probes One-Step kit for RT-qPCR amplification of the SARS-CoV-2 ORF1ab and N gene targets and quantified for SARS-CoV-2 genome equivalent copies (GEC) per mL of sample. None of the six hospitals had a primary wastewater treatment facility; two COVID hospitals had functional septic tanks, and the rest of the hospitals had either broken onsite systems or no containment of wastewater. Overall, 100 % of wastewater samples were positive with a high concentration of E. coli (mean = 7.0 log10 CFU/100 mL). Overall, 67 % (60/90) samples were positive for SARS-CoV-2. The highest SARS-CoV-2 concentrations (median: 141 GEC/mL; range: 13–18,214) were detected in wastewater from COVID-hospitals, and in non-COVID-hospitals, the median SARS-CoV-2 concentration was 108 GEC/mL (range: 30–1829). Our results indicate that high concentrations of E. coli and SARS-CoV-2 were discharged through the hospital wastewater (both COVID and non-COVID) without treatment into the ambient water bodies. Although there is no evidence for transmission of SARS-CoV-2 via wastewater, this study highlights the significant risk posed by wastewater from health care facilities in Dhaka for the many other diseases that are spread via faecal oral route. Hospitals in low-income settings could function as sentinel sites to monitor outbreaks through wastewater-based epidemiological surveillance systems. Hospitals should aim to adopt the appropriate wastewater treatment technologies to reduce the discharge of pathogens into the environment and mitigate environmental exposures.

Cross-Contamination to Surfaces in Consumer Kitchens with MS2 as a Tracer Organism in Ground Turkey Patties

It is estimated that one in five cases of foodborne illnesses is acquired in the home. However, how pathogens move throughout a kitchen environment when consumers are preparing food is not well characterized. The purpose of this study was to determine the prevalence and degree of cross-contamination across a variety of kitchen surfaces during a consumer meal preparation event. Consumers (n = 371) prepared a meal consisting of turkey patties containing the bacteriophage MS2 as a tracer organism and a ready-to-eat lettuce salad. Half were shown a video on proper thermometer use before the trial. After meal preparation, environmental sampling and detection were performed to assess cross-contamination with MS2. For most surfaces, positivity did not exceed 20%, with the exception of spice containers, for which 48% of the samples showed evidence of MS2 cross-contamination. Spice containers also had the highest MS2 concentrations, at a mean exceeding 6 log viral genome equivalent copies per surface. The high level of MS2 on spice containers drove the significant differences between surfaces, suggesting the significance of spice containers as a vehicle for cross-contamination, despite the absence of previous reports to this effect. The thermometer safety intervention did not affect cross-contamination. The efficiency of MS2 transfer, when expressed as a percentage, was relatively low, ranging from an average of 0.002 to 0.07%. Quantitative risk assessment work using these data would aid in further understanding the significance of cross-contamination frequency and efficiency. Overall, these data will help create more targeted consumer messaging to better influence consumer cross-contamination behaviors.

Comparative Assessment of the Efficacy of Commercial Hand Sanitizers Against Human Norovirus Evaluated by an in vivo Fingerpad Method.

Human noroviruses (hNoV) are the leading cause of acute non-bacterial gastroenteritis worldwide and contaminated hands play a significant role in the spread of disease. Some hand sanitizers claim to interrupt hNoV transmission, but their antiviral efficacy on human hands is poorly characterized. The purpose of this work was to characterize the efficacy of representative commercial hand sanitizers against hNoV using an in vivo fingerpad method (ASTM E1838-17). Eight products [seven ethanol-based and one benzalkonium chloride (BAK)-based], and a benchmark 60% ethanol solution, were each evaluated on 10 human volunteers using the epidemic GII.4 hNoV strain. Virus titers before and after treatment were evaluated by RT-qPCR preceded by RNase treatment; product efficacy was characterized by log10 reduction (LR) in hNoV genome equivalent copies after treatment. The benchmark treatment produced a 1.7 ± 0.5 LR, compared with Product A (containing 85% ethanol) which produced a 3.3 ± 0.3 LR and was the most efficacious (p < 0.05). Product B (containing 70% ethanol), while less efficacious than Product A (p < 0.05), performed better than the benchmark with a LR of 2.4 ± 0.4. Five of the other ethanol-based products (labeled ethanol concentration ranges of 62–80%) showed similar efficacy to the 60% ethanol benchmark with LR ranging from 1.3 to 2.0 (p > 0.05). Product H (0.1% BAK) was less effective than the benchmark with a LR of 0.3 ± 0.2 (p < 0.05). None of the products screened were able to completely eliminate hNoV (maximum assay resolution 5.0 LR). Product performance was variable and appears driven by overall formulation. There remains a need for more hand sanitizer formulations having greater activity against hNoV, a virus that is comparatively recalcitrant relative to other pathogens of concern in community, healthcare, and food preparation environments.

Simultaneous detection of Zika, chikungunya, dengue, yellow fever, West Nile, and Japanese encephalitis viruses by a two-tube multiplex real-time RT-PCR assay.

Due to the concurrent prevalence and increasing risk of coinfection of the clinically important Arboviruses, timely and accurate differential diagnosis is important for clinical management and the epidemiological investigation. A two-tube multiplex real-time reverse transcription-polymerase chain reaction (RT-PCR) assay for the simultaneous detection of Zika virus (ZIKV), chikungunya virus (CHIKV), dengue virus (DENV), yellow fever virus (YFV), West Nile virus (WNV), and Japanese encephalitis virus (JEV) was developed and optimized with high specificity and sensitivity. The detection limit for all the sixviruses could reach as low as fivegenome equivalent copies and 2.8 × 10-3 tissue culture infectious doses (TCID50 ) for ZIKV, YFV, CHIKV and 2.8 × 10-2 TCID50 for JEV per reaction, with high accuracy and precision (R2 > 0.99). The coefficient of variation of intra-assay and inter-assay for our quantitative reverse transcription-polymerase chain reaction (qRT-PCR) assay was low, and the obtained positive rates ad Ct values of this assay were comparable with singleplex commercial kits. Moreover, the multiplex qRT-PCR assay was able to detect possible co-infections without competitive inhibition of target viral genomes. In conclusion, our rapid, sensitive, cost-effective multiplex qRT-PCR will be of great use for differential diagnosis in a clinical setting and epidemiological investigation during surveillance.

Dose-Response of a Norovirus GII.2 Controlled Human Challenge Model Inoculum.

Genogroup II noroviruses are the most common cause of acute infectious gastroenteritis. We evaluated the use of a new GII.2 inoculum in a human challenge. Forty-four healthy adults (36 secretor-positive and 8 secretor-negative for histo-blood group antigens) were challenged with ascending doses of a new safety-tested Snow Mountain virus (SMV) GII.2 norovirus inoculum (1.2 × 104 to 1.2 × 107 genome equivalent copies [GEC]; n = 38) or placebo (n = 6). Illness was defined as diarrhea and/or vomiting postchallenge in subjects with evidence of infection (defined as GII.2 norovirus RNA detection in stool and/or anti-SMV immunoglobulin G [IgG] seroconversion). The highest dose was associated with SMV infection in 90%, and illness in 70% of subjects with 10 of 12 secretor-positive (83%) and 4 of 8 secretor-negative (50%) becoming ill. There was no association between prechallenge anti-SMV serum IgG concentration, carbohydrate-binding blockade antibody, or salivary immunoglobulin A and infection. The median infectious dose (ID50) was 5.1 × 105 GEC. High rates of infection and illness were observed in both secretor-positive and secretor-negative subjects in this challenge study. However, a high dose will be required to achieve the target of 75% illness to make this an efficient model for evaluating potential norovirus vaccines and therapeutics. NCT02473224.

Development of a Multiplex Droplet Digital PCR Assay for the Detection of Babesia, Bartonella, and Borrelia Species.

We describe the development, optimization, and validation of a multiplex droplet digital PCR (ddPCR) assay for the simultaneous detection of Babesia, Bartonella, and Borrelia spp. DNA from several sample matrices, including clinical blood samples from animals and humans, vectors, in-vitro infected human and animal cell lines, and tissues obtained from animal models (infected with Bartonella and/or B. burgdorferi). The multiplex ddPCR assay was able to detect 31 Bartonella, 13 Borrelia, and 24 Babesia species, including Theileria equi, T. cervi, and Cytauxzoon felis. No amplification of Treponema or Leptospira spp. was observed. Sensitivity of 0.2–5 genome equivalent DNA copies per microliter was achieved for different members of the Bartonella and Borrelia genus, depending on the species or matrix type (water or spiked blood DNA) tested. The ddPCR assay facilitated the simultaneous detection of co-infections with two and three vector-borne pathogens comprising four different genera (Babesia, Bartonella, Borrelia, and Theileria) from clinical and other sample sources.

Pigs Immunized with the Virus-like Particle Vaccine Are Protected against the Hepatitis E-3 Virus.

In this study, we generated the HEV virus-like particle (VLP) vaccine expressing 239 amino acids (367–605 aa) of the HEV-3 ORF2 using the baculovirus expression system. The HEV-3-239-VLP vaccine efficacy was evaluated by dividing 12 pathogen-free pigs into four groups: negative control, positive control, 100 μg VLP-, and 200 μg VLP-vaccinated groups for 10 weeks. The pigs in either of the vaccinated groups were administered the corresponding first and booster doses on weeks 0 and 2. At week 4, the positive control and two vaccinated groups were challenged with 106 HEV-3 genomic equivalent copies; viremia and fecal shedding of the virus were identified in pigs in the positive control and 100 μg VLP-vaccinated pigs showed transient viremia and fecal viral shedding. However, no viruses were detected in the serum or fecal samples of the 200 μg VLP-vaccinated pigs. The 100 and 200 μg VLP-vaccinated pigs had significantly higher (p < 0.01) anti-HEV antibodies than the negative control pigs from weeks 6–10 with normal levels of liver enzymes. The 200 μg VLP-vaccinated pigs showed statistically less liver tissue fibrosis (p < 0.05) than that of the positive control pigs. Thus, the novel baculovirus expression system-generated VLP vaccine dose-dependently protects against HEV-3 challenge and may be useful in other animal species, including humans.

Efficacy of alcohol-based hand sanitizers against human norovirus using RNase-RT-qPCR with validation by human intestinal enteroid replication.

Successful human norovirus (HuNoV) cultivation in stem cell‐derived human intestinal enteroids (HIE) was recently reported. The purpose of this study was to evaluate the anti‐HuNoV efficacy of two alcohol‐based commercial hand sanitizers and 60% ethanol by suspension assay using RNase‐RT‐qPCR, with subsequent validation of efficacy by HuNoV cultivation using the HIE model. In suspension, when evaluated by RNase‐RT‐qPCR, 60% ethanol resulted in less than one log10 reduction in HuNoV genome equivalent copies (GEC) regardless of contact time (30 or 60s) or soil load. The two commercial products outperformed 60% ethanol regardless of contact time or soil load, providing 2·2–3·2 log10 HuNoV GEC reductions by suspension assay. Product B could not be validated in the HIE model due to cytotoxicity. Following a 60s exposure, viral replication in the HIE model increased 1·9 ± 0·2 log10 HuNoV GEC for the neutralization (positive) control and increased 0·9 ± 0·2 log10 HuNoV GEC in challenged HIE after treatment with 60% ethanol. No HuNoV replication in HIE was observed after a 60 s exposure to Product A.

Development of an RT-LAMP assay for the detection of Lassa viruses in southeast and south-central Nigeria

Lassa virus (LASV) causes Lassa fever (LF), a viral hemorrhagic fever endemic in West Africa. LASV strains are clustered into six lineages according to their geographic location. To confirm a diagnosis of LF, a laboratory test is required. Here, a reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay using a portable device for the detection of LASV in southeast and south-central Nigeria using three primer sets specific for strains clustered in lineage II was developed. The assay detected in vitro transcribed LASV RNAs within 23 min and was further evaluated for detection in 73 plasma collected from suspected LF patients admitted into two health settings in southern Nigeria. The clinical evaluation using the conventional RT-PCR as the reference test revealed a sensitivity of 50% in general with 100% for samples with a viral titer of 9500 genome equivalent copies (geq)/mL and higher. The detection limit was estimated to be 4214 geq/mL. The assay showed 98% specificity with no cross-reactivity to other viruses which cause similar symptoms. These results suggest that this RT-LAMP assay is a useful molecular diagnostic test for LF during the acute phase, contributing to early patient management, while using a convenient device for field deployment and in resource-poor settings.

Development of a quadruple qRT-PCR assay for simultaneous identification of highly and low pathogenic H7N9 avian influenza viruses and characterization against oseltamivir resistance

BackgroundDuring the fifth wave of human H7N9 infections, a novel highly pathogenic (HP) H7N9 variant emerged with an insertion of multiple basic amino acids in the HA cleavage site. Moreover, a neuraminidase inhibitor (NAI) resistance (R292K in NA) mutation was found in H7N9 isolates from humans, poultry and the environment. In this study, we set out to develop and validate a multiplex quantitative reverse transcript polymerase chain reaction (qRT-PCR) to simultaneously detect the presence of H7N9 and further identify the HP and NAI-resistance mutations.MethodsA quadruple qRT-PCR to simultaneously detect the presence of H7N9 and further identify the HP and NAI-resistance mutations was designed based on the analyses of the HA and NA genes of H7N9. This assay was further tested for specificity and sensitivity, and validated using clinical samples.ResultsThe assay was highly specific and able to detect low pathogenic (LP)- or HP-H7N9 with/without the NAI-resistance mutation. The detection limit of the assay was determined to be 50 genome-equivalent copies and 2.8 × 10− 3 50% tissue culture infectious doses (TCID50) of live H7N9 per reaction. Clinical validation was confirmed by commercial kits and Sanger sequencing with ten clinical samples.ConclusionsWe developed and validated a rapid, single-reaction, one-step, quadruple real-time qRT-PCR to simultaneously detect the presence of H7N9 and further identify the HP- and NAI-resistance strains with excellent performance in specificity and sensitivity. This assay could be used to monitor the evolution of H7N9 viruses in the laboratory, field and the clinic for early-warning and the prevention of H7N9 infections.

PD56-03 DETECTION OF PRIVATE CLONAL MUTATIONS IN LOCALISED PROSTATE CANCER FROM RARE MOLECULES OF CIRCULATING TUMOUR DNA

You have accessJournal of UrologyProstate Cancer: Markers II1 Apr 2018PD56-03 DETECTION OF PRIVATE CLONAL MUTATIONS IN LOCALISED PROSTATE CANCER FROM RARE MOLECULES OF CIRCULATING TUMOUR DNA Keval Patel, Charlie Massie, Francesco Marass, James Morris, Andy Lynch, Florent Mouliere, David Neal, Vincent Gnanapragasam, Tim Forshew, and Nitzan Rosenfeld Keval PatelKeval Patel More articles by this author , Charlie MassieCharlie Massie More articles by this author , Francesco MarassFrancesco Marass More articles by this author , James MorrisJames Morris More articles by this author , Andy LynchAndy Lynch More articles by this author , Florent MouliereFlorent Mouliere More articles by this author , David NealDavid Neal More articles by this author , Vincent GnanapragasamVincent Gnanapragasam More articles by this author , Tim ForshewTim Forshew More articles by this author , and Nitzan RosenfeldNitzan Rosenfeld More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2018.02.2635AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Prostate cancer (PCa) is the most common solid tumour in men. Current methods of distinguishing between indolent and aggressive PCa can be inaccurate due to PCa heterogeneity and multi-focality. To compensate, many centres now take upwards of 20 biopsies of the prostate in one sitting, and monitor men with further biopsies to ensure that the disease is not progressing. There is therefore a need for a non-invasive biomarker than can monitor tumour heterogeneity and aggression. Circulating-tumour DNA (ctDNA) represents an exciting opportunity to monitor cancer status non-invasively. We developed a method for analysis of multiple genomic regions whilst retaining sensitivity for detection of individual mutant molecules, using multiplex replicate dilution sequencing, or MRD-Seq. METHODS MRD-Seq distributes a DNA sample into multiple replicate PCR reactions, where each reaction contains a small number of initial template molecules. As a result, the detection of a single mutant molecule generates a signal that is clearly discernible above the background noise. Dilutions series determined the limit of detection (0.09% AF) and background noise (0.02%AF). To assess ctDNA detection in the context of intra-tumoural heterogeneity, prostatectomy specimens and pre-operative plasma was obtained from 2 men with localised but multifocal PCa, with appropriate ethical approval (MREC:01/4/061). Whole genome sequencing of spatially 5-7 distinct regions of prostate cancer for each man was used to design probes to interrogate private mutations. RESULTS Plasma MRD-Seq demonstrated a mutant AF of 0.9% and 0.4% in the 2 men from ~1,000 genomic equivalent copies input. MRD-Seq detected the presence of private mutations from multiple private clones in the plasma, with evidence that more aggressive tumour regions (according to Gleason Grade) were over-represented in the plasma. CONCLUSIONS This proof of principle study suggests that ctDNA is detectable in the plasma of localised PCa patients and could be used to monitor men on active surveillance and identify aggressive changes at an early stage. © 2018FiguresReferencesRelatedDetails Volume 199Issue 4SApril 2018Page: e1062-e1063 Advertisement Copyright & Permissions© 2018MetricsAuthor Information Keval Patel More articles by this author Charlie Massie More articles by this author Francesco Marass More articles by this author James Morris More articles by this author Andy Lynch More articles by this author Florent Mouliere More articles by this author David Neal More articles by this author Vincent Gnanapragasam More articles by this author Tim Forshew More articles by this author Nitzan Rosenfeld More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Screening of rice (Oryza sativa) cultivars for resistance to rice black streaked dwarf virus using quantitative PCR and visual disease assessment

Rice black streaked dwarf virus (RBSDV) causing rice black streaked dwarf disease is transmitted by the small brown planthopper (SBPH, Laodelphax striatellus) in a persistent propagative manner. The disease is considered among the most destructive in rice production in east and southeast Asia. For sustainable control of the disease, planting resistant cultivars is the most economical and efficient method. The virus content in different rice cultivars was quantified using a TaqMan RT‐qPCR assay under greenhouse conditions and the disease was visually assessed in these cultivars in both greenhouse and field conditions. Results revealed significant positive moderate correlation (r = 0.3787; P = 0.0009) between the virus content and visual disease assessment in the greenhouse under forced inoculation. Among 66 rice cultivars, there was no significant difference in RBSDV genome equivalent copies (GEC) in seven cultivars, namely Lian‐dao 9805 (200.2 ± 12), Liangyou 3399 (206.6 ± 28), Ningjing 4 (206.6 ± 28), DaLiang 207 (302.0 ± 61), X 008 (354.0 ± 30), Shengdao 301 (658.4 ± 69) and Liangyou 1129 (679.5 ± 98). These cultivars were also visually assessed as resistant under greenhouse and field conditions. These cultivars could be used in disease management to reduce the likelihood of epidemics.

Vomiting as a Symptom and Transmission Risk in Norovirus Illness: Evidence from Human Challenge Studies.

BackgroundIn the US, noroviruses are estimated to cause 21 million cases annually with economic losses reaching $2 billion. Outbreak investigations frequently implicate vomiting as a major transmission risk. However, little is known about the characteristics of vomiting as a symptom or the amount of virus present in emesis.Methodology and Principal FindingsEmesis samples and symptomology data were obtained from previous norovirus human challenge studies with GI.1 Norwalk virus, GII.2 Snow Mountain virus, and a pilot study with GII.1 Hawaii virus. Viral titers in emesis were determined using strain-specific quantitative RT-PCR. In all four studies, vomiting was common with 40–100% of infected subjects vomiting at least once. However, only 45% of subjects with vomiting also had diarrhea. Most of the emesis samples had detectable virus and the mean viral titers were 8.0 x 105 and 3.9 x 104 genomic equivalent copies (GEC)/ml for GI and GII viruses, respectively (p = 0.02). Sample pH was correlated with GII.2 Snow Mountain virus detection.Conclusions and SignificanceHalf of all subjects with symptomatic infection experienced vomiting and the average subject shed 1.7 x 108 GEC in emesis. Unlike shedding through stool, vomiting is more likely to result in significant environmental contamination, leading to transmission through fomites and airborne droplets. This quantitative data will be critical for risk assessment studies to further understand norovirus transmission and develop effective control measures. The correlation between sample pH and virus detection is consistent with a single site of virus replication in the small intestine and stomach contents becoming contaminated by intestinal reflux. Additionally, the frequency of vomiting without concurrent diarrhea suggests that epidemiology studies that enroll subjects based on the presence of diarrhea may be significantly underestimating the true burden of norovirus disease.

Development of an Orientia tsutsugamushi Lc-1 Murine Intraperitoneal Challenge Model for Scrub Typhus: Determination of Murine Lethal Dose (MuLD50), Tissue Bacterial Loads, and Clinical Outcomes.

Currently, no vaccine has been developed to protect humans from naturally acquired heterologous Orientia tsutsugamushi infections. To enhance the validity of vaccine candidates, we are developing a murine chigger challenge model with the O. tsutsugamushi Lc-1-infected Leptotrombidium chiangraiensis Line-1. To this end, an intraperitoneal (i.p.) murine challenge model using an O. tsutsugamushi Lc-1 isolate was developed for eventual validation of the chigger challenge model. We have determined that the murine lethal dose that kills 50% of the challenged mice (MuLD50) of a liver/spleen homogenate developed from O. tsutsugamushi Lc-1-infected ICR Swiss mice to be 10(-6.9). Employing different inoculum doses of this homogenate, the bacterial load using quantitative real-time PCR (qPCR) was determined to range from 60 to 1.6 × 10(5) genome equivalent copies (GEC)/μL of liver and 33.4 to 2.2 × 10(5) GEC/μL of spleen tissue. The clinical outcomes relative to homogenate dose levels followed a dose-dependent pattern. The successful development and characterization of the O. tsutsugamushi Lc-1 i.p. challenge model will assist in the development and validation of a mouse chigger challenge scrub typhus model.

Quantification of southern rice black streaked dwarf virus and rice black streaked dwarf virus in the organs of their vector and nonvector insect over time

Southern rice black streaked dwarf virus (SRBSDV) and rice black streaked dwarf virus (RBSDV) are serious rice-infecting reoviruses, which are transmitted by different planthoppers in a persistent propagative manner. In this study, we quantitatively compared the spatial distribution of SRBSDV and RBSDV contents over time in their vector and nonvector insects using real time-PCR. Genome equivalent copies (GEC) were assessed every 2 days from 0 to 14 days after a 3-days acquisition access period (AAP) on infected plants. Results revealed 293.2±21.6 to 404.1±46.4 SRBSDV GEC/ng total RNA in whole body of white-backed planthopper (WBPH, Sogatella furcifera) at day 0 and 12 and 513.5±88.4 to 816.8±110.7 RBSDV GEC/ng total RNA in the whole body of small brown planthopper (SBPH, Laodelphax striatellus) at day 0 and 14, respectively, after 3-days AAP. Highest GEC of both viruses were found in the gut of their respective vectors. Although SRBSDV was detected in the gut of SBPH, it did not spread into the hemolymph or other organs. After an 8-day latent period, the transmission efficiency of SRBSDV and RBSDV by their respective vectors was significantly positively correlated with GEC in the salivary gland (r2=0.7808, P=0.0036 and r2=0.9351, P<0.0001, respectively, at α=0.05). Together, these results confirm that accumulation of >200 SRBSDV or RBSDV GEC/ng total RNA in the gut of vector, indicated threshold for further spread and the virus content in the salivary gland was significantly correlated with transmission efficiency by their respective vectors.

Comparison of process control viruses for use in extraction and detection of human norovirus from food matrices

Although RT-qPCR is a powerful tool for human norovirus (HuNoV) detection, low virus concentrations in potentially large sample volumes necessitate the use of inefficient sample processing step(s) prior to detection. Process control viruses (PCVs) are used to monitor the efficiency of these virus concentration steps. This study compared five PCVs [Mengovirus (Mengo), murine norovirus (MNV-1), MS2 coliphage, Tulane virus, and turnip crinkle virus (TCV)] to two HuNoV strains for recovery during the steps of elution, polyethylene glycol precipitation (PEG), and RNA extraction from select foods (lettuce and sliced deli ham). Results demonstrate high recovery efficiencies of HuNoV GI.6 and GII.4 using the methods described in this study: combined (sequential) losses during processing from sliced deli ham and lettuce were <1 log10 genome equivalent copies (GEC). When considering the processing steps separately, HuNoV loss was negligible after elution, and low after PEG precipitation (mean 0.5 log10 GEC) and RNA extraction (mean 0.1 log10 GEC). The virus that least mimicked the behavior of HuNoV during sample processing was MNV-1. Of the viruses tested, a commercial mengovirus strain gave recovery efficiencies closest to HuNoV, showing combined losses from sliced deli ham and lettuce of <1 log10 GEC and ~1 log10 GEC, respectively. All PCVs do not behave equivalently and validation of their performance is recommended before their routine use on an application-by-application basis.