The Three-Cornered Alfalfa Hopper, Spissistilus festinus, Is a Vector of Grapevine Red Blotch Virus in Vineyards.

Spissistilus festinus (Hemiptera: Membracidae) transmit grapevine red blotch virus (GRBV, Grablovirus, Geminiviridae) in greenhouse settings; however, their role as a vector of GRBV in vineyards is unknown. Following controlled exposures of aviruliferous S. festinus for two weeks on infected, asymptomatic vines in a California vineyard in June and a 48 h gut clearing on alfalfa, a nonhost of GRBV, approximately half of the released insects tested positive for GRBV (45%, 46 of 102), including in the salivary glands of dissected individuals (11%, 3 of 27), indicating acquisition. Following controlled exposures of viruliferous S. festinus for two to six weeks on GRBV-negative vines in vineyards in California and New York in June, transmission of GRBV was detected when two S. festinus were restricted to a single leaf (3%, 2 of 62 in California; 10%, 5 of 50 in New York) but not with cohorts of 10-20 specimens on entire or half shoots. This work was consistent with greenhouse assays in which transmission was most successful with S. festinus exposed to a single leaf (42%, 5 of 12), but rarely occurred on half shoots (8%, 1 of 13), and never on entire shoots (0%, 0 of 18), documenting that the transmission of GRBV is facilitated through the feeding of fewer S. festinus on a restricted area of grapevine tissue. This work demonstrates S. festinus is a GRBV vector of epidemiological importance in vineyards.

Complete Genome Sequence Resource for a Recently Isolated Potato Ring Rot Pathogen, Clavibacter sepedonicus K496.

Potato ring rot caused by Clavibacter sepedonicus has been a devastating disease in the U.S. since 1930. In this study, we isolated a recent C. sepedonicus strain, K496, from potato tubers showing discolorations of the vascular cylinder or pith tissues. We de novo assembled the genome sequence of K496 with 1,924,544,313 bp of Nanopore reads (N50 = 13,785 bp) using Flye v2.9 and polished it with 2 × 150 bp paired-end Illumina reads (855,788,703 bp in total). The resulting genome of K496 consists of a single circular chromosome 3,266,016 bp long and a linear plasmid of 135,489 bp. Using the NCBI PGAP v5.3, this genome was predicted to have 3,301 genes, encompassing 3,247 protein-coding genes, 90 pseudogenes, two 5S rRNA-coding, two 16S rRNA-coding, two 23S rRNA-coding sequences, 45 tRNAs, and three noncoding RNAs. The chromosome and plasmid sequences have been deposited at the NCBI GenBank database under the accession numbers CP088266 and CP088267, respectively.

Psychosocial assessment tools for use before transplantation are predictive of post-operative psychosocial and health behavior outcomes: a narrative review of the literature.

Introduction: In end-stage diseases, transplantation may be necessary. The limited number of donors led to the development of several pre-transplant psychosocial assessment tools. We summarized the predictive value of these tools before solid-organ transplantation. Methods: The PRISMA search strategy and the MEDLINE database were used to review the literature. From 1,050 records, we found thirteen studies using four different scales (Millon Behavioral Health Inventory [MBHI], Psychosocial Assessment of Transplant Candidates [PACT], Stanford Integrated Psychosocial Assessment for Transplantation [SIPAT], and Transplant Evaluation Rating Scale [TERS]). Results: TERS and MBHI were associated with the highest number of positive studies concerning pre-transplant scores and primary outcomes. Psychosocial scales predict in a systematic way psychosocial and health behavioural outcomes, but generated mixed results for mortality and rejection. Discussion: This narrative review underlines the need for multidisciplinary evaluation and well-conducted clinical trials to assist transplant teams in utilizing psychosocial evaluation effectively during evaluation of candidates.

Transmission of Grapevine Red Blotch Virus by Spissistilus festinus [Say, 1830] (Hemiptera: Membracidae) between Free-Living Vines and Vitis vinifera 'Cabernet Franc'.

Grapevine red blotch disease emerged within the past decade, disrupting North American vine stock production and vineyard profitability. Our understanding of how grapevine red blotch virus (GRBV), the causal agent of the disease, interacts with its Vitis hosts and insect vector, Spissistilus festinus, is limited. Here, we studied the capabilities of S. festinus to transmit GRBV from and to free-living vines, identified as first-generation hybrids of V. californica and V. vinifera 'Sauvignon blanc' (Vcal hybrids), and to and from V. vinifera 'Cabernet franc' (Vvin Cf) vines. The transmission rate of GRBV was high from infected Vcal hybrid vines to healthy Vcal hybrid vines (77%, 10 of 13) and from infected Vvin Cf vines to healthy Vcal hybrid vines (100%, 3 of 3). In contrast, the transmission rate of GRBV was low from infected Vcal hybrid vines to healthy Vvin Cf vines (15%, 2 of 13), and from infected Vvin Cf vines to healthy Vvin Cf vines (19%, 5 of 27). No association was found between transmission rates and GRBV titer in donor vines used in transmission assays, but the virus titer was higher in the recipient leaves of Vcal hybrid vines compared with recipient leaves of Vvin Cf vines. The transmission of GRBV from infected Vcal hybrid vines was also determined to be trans-stadial. Altogether, our findings revealed that free-living vines can be a source for the GRBV inoculum that is transmissible by S. festinus to other free-living vines and a wine grape cultivar, illustrating the interconnected roles of the two virus hosts in riparian areas and commercial vineyards, respectively, for virus spread. These new insights into red blotch disease epidemiology will inform the implementation of disease management strategies.

Species of Dickeya and Pectobacterium Isolated during an Outbreak of Blackleg and Soft Rot of Potato in Northeastern and North Central United States.

An outbreak of bacterial soft rot and blackleg of potato has occurred since 2014 with the epicenter being in the northeastern region of the United States. Multiple species of Pectobacterium and Dickeya are causal agents, resulting in losses to commercial and seed potato production over the past decade in the Northeastern and North Central United States. To clarify the pathogen present at the outset of the epidemic in 2015 and 2016, a phylogenetic study was made of 121 pectolytic soft rot bacteria isolated from symptomatic potato; also included were 27 type strains of Dickeya and Pectobacterium species, and 47 historic reference strains. Phylogenetic trees constructed based on multilocus sequence alignments of concatenated dnaJ, dnaX and gyrB fragments revealed the epidemic isolates to cluster with type strains of D. chrysanthemi, D. dianthicola, D. dadantii, P. atrosepticum, P. brasiliense, P. carotovorum, P. parmentieri, P. polaris, P. punjabense, and P. versatile. Genetic diversity within D. dianthicola strains was low, with one sequence type (ST1) identified in 17 of 19 strains. Pectobacterium parmentieri was more diverse, with ten sequence types detected among 37 of the 2015-2016 strains. This study can aid in monitoring future shifts in potato soft rot pathogens within the U.S. and inform strategies for disease management.

Accuracy of four lateral flow immunoassays for anti SARS-CoV-2 antibodies: a head-to-head comparative study.

BACKGROUND: SARS-CoV-2 antibody tests are used for population surveillance and might have a future role in individual risk assessment. Lateral flow immunoassays (LFIAs) can deliver results rapidly and at scale, but have widely varying accuracy. METHODS: In a laboratory setting, we performed head-to-head comparisons of four LFIAs: the Rapid Test Consortium's AbC-19TM Rapid Test, OrientGene COVID IgG/IgM Rapid Test Cassette, SureScreen COVID-19 Rapid Test Cassette, and Biomerica COVID-19 IgG/IgM Rapid Test. We analysed blood samples from 2,847 key workers and 1,995 pre-pandemic blood donors with all four devices. FINDINGS: We observed a clear trade-off between sensitivity and specificity: the IgG band of the SureScreen device and the AbC-19TM device had higher specificities but OrientGene and Biomerica higher sensitivities. Based on analysis of pre-pandemic samples, SureScreen IgG band had the highest specificity (98.9%, 95% confidence interval 98.3 to 99.3%), which translated to the highest positive predictive value across any pre-test probability: for example, 95.1% (95% uncertainty interval 92.6, 96.8%) at 20% pre-test probability. All four devices showed higher sensitivity at higher antibody concentrations ("spectrum effects"), but the extent of this varied by device. INTERPRETATION: The estimates of sensitivity and specificity can be used to adjust for test error rates when using these devices to estimate the prevalence of antibody. If tests were used to determine whether an individual has SARS-CoV-2 antibodies, in an example scenario in which 20% of individuals have antibodies we estimate around 5% of positive results on the most specific device would be false positives. FUNDING: Public Health England.

An anomalous detection of Tomato yellow leaf curl virus in tomato in New York State.

In August 2020, a New York State vegetable grower sought assistance to identify a malady of tomato (Solanum lycopersicum). The plants were grown from saved seed that had been planted annually in NY and/or FL for over 15 years without significant disease problems, but the identity of the cultivar was not known. Submitted photos showed severely stunted plants with distorted leaves (crinkling, cupping, twisting); leaves were reduced in size and showed interveinal yellowing. Although the most likely explanation given the growing region was herbicide damage, the symptoms bore a striking resemblance to those presented by tomato yellow leaf curl (TYLCV)-infected tomato plants. TYLCV has not been reported from NY, as the whitefly vector (Bemisia tabaci) does not overwinter in the region. Stem tissue from a symptomatic plant was grafted onto a greenhouse grown rootstock of tomato breeding line 201231 (Cornell University); shoots emerging from grafted rootstocks showed symptoms consistent with those on the scion within 21 days of grafting. Total nucleic acid was extracted (Gambino et al. 2008), and a polymerase chain reaction (PCR) assay to detect TYLCV was performed using primers AV632 and AC1048 (Martínez-Culebras et al. 2001). Sanger sequencing of the expected size ~460 bp product from a representative sample showed 98% nucleotide identity with the sequence of over 52 isolates of TYLCV (blastn analysis using default parameters; Altschul et al. 1990). The total nucleic acid preparation was subjected to rolling circle ampification followed by restriction enzyme SphI digestion (Haible et al. 2006). An approximately 2.8 kb DNA fragment was resolved by agarose gel electrophoresis, gel purified, inserted into the cloning vector pUC19 and sequenced. Two clones yielded sequence of 2781 nt with only one nt mismatch (accession # MW373746, MW373747). BLAST analysis showed the sequence to be most closely related to TYLCV-IL from papaya in Texas (accession KX024647.1) with 99% identity (2752 of 2781 nt). Further inquiry revealed that the vegetable grower's plants had been seeded and grown in Florida prior to transplanting in NY; Florida is a production region where the virus and vectors are endemic. Although the virus has been shown to be associated with seed (Pérez-Padilla et al. 2020) and seed transmission has been reported (Kil et al. 2016), this subject is controversial and the epidemiology of the disease is not consistent with a seed-transmitted virus (Rojas, et al. 2018). In this reported occurrence, the most plausible explanation is that the virus was introduced into NY with transplants. All of the field grown transplants of this cultivar were infected, but no local disease spread in NY was reported, nor were there reports of the vector. The significance of this report is to highlight the importance of phytosanitation in the movement of plants and plant materials. The long-distance movement of TYLCV via infected transplants in the US and globally is well-established. The presence of a pathogen may be transient and their establishment will depend on the epidemiology of the pathogen, in this case, the presence of the vector.

Robust antibody responses in 70-80-year-olds 3 weeks after the first or second doses of Pfizer/BioNTech COVID-19 vaccine, United Kingdom, January to February 2021.

Sera were collected from 185 adults aged ≥ 70 years in London to evaluate the immune response to COVID-19 vaccines. A single dose of Pfizer/BioNtech vaccine resulted in > 94% seropositivity after 3 weeks in naïve individuals using the Roche Spike antibody assay, while two doses produced very high spike antibody levels, significantly higher than convalescent sera from mild-to-moderate PCR-confirmed adult cases. Our findings support the United Kingdom's approach of prioritising the first dose and delaying the second dose of COVID-19 vaccine.

Grapevine Red Blotch Virus Is Transmitted by the Three-Cornered Alfalfa Hopper in a Circulative, Nonpropagative Mode with Unique Attributes.

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 after 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 nonhost of GRBV, the virus titer decreased over time in adult insects, as shown by quantitative PCR. Snap bean proved to be a feeding host of S. festinus and a pseudosystemic host of GRBV after 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 trans-stadially but not via seeds. The virus titer was significantly higher in (i) guts and hemolymph relative to heads with salivary glands, and (ii) 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.

Cultivated and Wild Grapevines in Tennessee Possess Overlapping but Distinct Virus Populations.

Viruses and viroids prevalent in a population of 42 wild grapevines (i.e., free-living, uncultivated grapevines; Vitis spp.) were compared with those in a population of 85 cultivated grapevines collected in Tennessee, United States by RNA sequencing analysis of pools of ribosomal RNA-depleted total RNA. The sequences of 10 viruses (grapevine fleck virus, grapevine leafroll-associated virus 2, grapevine rupestris stem pitting-associated virus, grapevine Syrah virus 1, grapevine vein-clearing virus, grapevine virus B, grapevine virus E, tobacco ringspot virus, tomato ringspot virus, and a novel nano-like virus) and two viroids (hop stunt viroid and grapevine yellow speckle viroid 1) were detected in both grapevine populations. Sequences of four viruses (grapevine associated tymo-like virus, grapevine leafroll-associated virus 3, grapevine red blotch virus, and grapevine virus H) were identified only from cultivated grapevines. High, moderate, and low numbers of sequence reads were identified only from wild grapevines for a novel caulimovirus, an enamovirus, and alfalfa mosaic virus, respectively. The presence of most virus sequences and both viroids was verified independently in the original samples by reverse-transcription PCR followed by Sanger sequencing. Comparison of viral sequences shared by both populations showed that cultivated and wild grapevines harbored distinct sequence variants, which suggests that there was limited virus movement between the two populations. Collectively, this study represents the first unbiased survey of viruses and viroids in both cultivated and wild grapevines within a defined geographic region.

Identification of Pectobacterium versatile Causing Blackleg of Potato in New York State.

Soft rot bacteria classified in the Pectobacteriaceae (SRP), including Pectobacterium and Dickeya spp., are responsible for soft rot and blackleg diseases of potato. Since 2014, blackleg outbreaks caused by D. dianthicola have increased in the United States and Canada. Our previous study found that the most abundant causal organisms of blackleg disease in New York State were P. parmentieri and D. dianthicola, with the latter being the only Dickeya species reported. In the present study, we identified and characterized pathogenic SRP bacteria from 19 potato samples collected in New York State during the 2017 growing season. We used genome sequence comparison to determine the pathogens' species. We found eight P. versatile, one P. atrosepticum, two P. carotovorum, two P. parmentieri, and six D. dianthicola isolates in our 2017 SRP collection. This is the first time that P. versatile has been reported to cause potato blackleg disease in New York State. We determined the phylogenetic relationships between the SRP strains by using 151 single-copy orthologous gene sequences shared among the set of bacteria in our analysis, which provided better resolution than phylogenies constructed with the dnaX gene.

Grapevine Asteroid Mosaic-Associated Virus is Resident and Prevalent in Wild, Noncultivated Grapevine of New York State.

In North America, uncultivated, free-living grapevines (Vitis spp.) frequently grow alongside their cultivated counterparts, thus increasing the potential for exchange of microbiota. For this study, we used high-throughput sequencing (HTS) of small RNAs to survey for virus populations in free-living grapevines of the Finger Lakes region of New York State. Of 32 grapevines analyzed, 23 were free-living vines, while the remaining 9 were commercially grown Vitis vinifera plants from the same region. In total, 18 (78.3%) of the free-living grapevines tested were positive for grapevine asteroid mosaic-associated virus (GAMaV) infection by HTS, with detection confirmed by seminested reverse-transcription PCR and sequencing of nine isolates. Phylogenetic analyses of an ungapped alignment of the New York GAMaV sequences (length: 2,334 nucleotides) with the five known full-length or close to full-length global sequences showed that the New York isolates were broadly grouped. Of the nine cultivated plants, eight were infected with both hop stunt viroid and grapevine yellow speckle viroid 1, three were singly infected with grapevine leafroll-associated virus 3, and one harbored GAMaV. This limited survey of free-living grapevines, one of the first to use HTS, has highlighted the high incidence of a virus associated with disease in commercial V. vinifera.

Accuracy of UK Rapid Test Consortium (UK-RTC) "AbC-19 Rapid Test" for detection of previous SARS-CoV-2 infection in key workers: test accuracy study.

OBJECTIVE: To assess the accuracy of the AbC-19 Rapid Test lateral flow immunoassay for the detection of previous severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. DESIGN: Test accuracy study. SETTING: Laboratory based evaluation. PARTICIPANTS: 2847 key workers (healthcare staff, fire and rescue officers, and police officers) in England in June 2020 (268 with a previous polymerase chain reaction (PCR) positive result (median 63 days previously), 2579 with unknown previous infection status); and 1995 pre-pandemic blood donors. MAIN OUTCOME MEASURES: AbC-19 sensitivity and specificity, estimated using known negative (pre-pandemic) and known positive (PCR confirmed) samples as reference standards and secondly using the Roche Elecsys anti-nucleoprotein assay, a highly sensitive laboratory immunoassay, as a reference standard in samples from key workers. RESULTS: Test result bands were often weak, with positive/negative discordance by three trained laboratory staff for 3.9% of devices. Using consensus readings, for known positive and negative samples sensitivity was 92.5% (95% confidence interval 88.8% to 95.1%) and specificity was 97.9% (97.2% to 98.4%). Using an immunoassay reference standard, sensitivity was 94.2% (90.7% to 96.5%) among PCR confirmed cases but 84.7% (80.6% to 88.1%) among other people with antibodies. This is consistent with AbC-19 being more sensitive when antibody concentrations are higher, as people with PCR confirmation tended to have more severe disease whereas only 62% (218/354) of seropositive participants had had symptoms. If 1 million key workers were tested with AbC-19 and 10% had actually been previously infected, 84 700 true positive and 18 900 false positive results would be projected. The probability that a positive result was correct would be 81.7% (76.8% to 85.8%). CONCLUSIONS: AbC-19 sensitivity was lower among unselected populations than among PCR confirmed cases of SARS-CoV-2, highlighting the scope for overestimation of assay performance in studies involving only PCR confirmed cases, owing to "spectrum bias." Assuming that 10% of the tested population have had SARS-CoV-2 infection, around one in five key workers testing positive with AbC-19 would be false positives. STUDY REGISTRATION: ISRCTN 56609224.

Complete Genome Sequence of a Gram-Positive Bacterium, Leifsonia sp. Strain PS1209, a Potato Endophyte.

We report the complete and annotated genome sequence of a Gram-positive bacterium, Leifsonia sp. strain PS1209, a potato endophyte that was isolated from apparently healthy tubers of potato cultivar NY166. The circular genome is 4,091,164 bp long, with a GC content of 69.08%, containing 3,926 genes.

A rapid, sensitive and inexpensive method for detection of grapevine red blotch virus without tissue extraction using loop-mediated isothermal amplification.

Grapevine red blotch virus (GRBV) is an emerging virus of significant viticultural importance throughout North America. Here, we report the development of a simple protocol for point-of-use detection of GRBV. Extraction of nucleic acids is not required; instead, the whole intact plant can simply be pricked with a sterile pipette tip, which is then incubated in sterile distilled water to provide the sample template in a loop-mediated isothermal amplification (LAMP) reaction. This method is 10,000 times more sensitive than conventional PCR, costs under a dollar per sample, and can be completed from sampling to readout in just over half an hour.

Complete Genome Sequence of Dickeya dianthicola ME23, a Pathogen Causing Blackleg and Soft Rot Diseases of Potato.

In 2014, an outbreak of potato blackleg and soft rot disease emerged in North America and continues to impact potato production. Here, we report the annotated genome sequence of Dickeya dianthicola ME23, a strain hypothesized to be representative of the bacterial population responsible for this disease outbreak.

Evidence for the splicing of grablovirus transcripts reveals a putative novel open reading frame.

Grapevine red blotch virus (GRBV) is type member of the newly identified genus Grablovirus. It possesses a single-stranded circular DNA genome of around 3200 nucleotides encoding three open reading frames (ORFs) in both the virion sense, the V1 (CP), V2 and V3, and complementary sense, C1 (RepA), C2 and C3. As shown for members of the genus Mastrevirus, the C1 and C2 ORFs are predicted to fuse through splicing to form a replication-associated protein (Rep). Data obtained using high-throughput sequencing (RNA-Seq) of three RNA-enriched populations, extracted from GRBV-infected grapevine (Vitis vinifera), confirmed the presence of the predicted C1-C2 intron (nts 2288-2450), but in addition identified a larger virion-sense intron (nts 251-589) spanning the V2 ORF. Evidence for both introns in a number of isolates was supported by bioinformatic analysis of publicly available datasets (n=20). These observations were further supported by RT-PCR analyses in both GRBV-infected grapevine and transient expression assays where GRBV genome segments were agro-inoculated onto Nicotiana benthamiana. The donor site of the virion-sense intron is located within two small ORFs, V0 and V02, while the acceptor site is two-thirds along the V2 ORF. Splicing at these positions is predicted to delete the N terminus of the encoded V2 protein. Comparative analyses of full-length GRBV sequences and the related tentative grabloviruses Prunus geminivirus A and wild Vitis virus 1 support the existence of both introns and V0. The probable regulatory role of these introns in the GRBV infection cycle is discussed.

A Small RNA-Mediated Regulatory Network in Arabidopsis thaliana Demonstrates Connectivity Between phasiRNA Regulatory Modules and Extensive Co-Regulation of Transcription by miRNAs and phasiRNAs.

Gene regulation involves the orchestrated action of multiple regulators to fine-tune the expression of genes. Hierarchical interactions and co-regulation among regulators are commonly observed in biological systems, leading to complex regulatory networks. Small RNA (sRNAs) have been shown to be important regulators of gene expression due to their involvement in multiple cellular processes. In plants, microRNA (miRNAs) and phased small interfering RNAs (phasiRNAs) correspond to two well-characterized types of sRNAs involved in the regulation of posttranscriptional gene expression, although information about their targets and interactions with other gene expression regulators is limited. We describe an extended sRNA-mediated regulatory network in Arabidopsis thaliana that provides a reference frame to understand sRNA biogenesis and activity at the genome-wide level. This regulatory network combines a comprehensive evaluation of phasiRNA production and sRNA targets supported by degradome data. The network includes ~17% of genes in the A. thaliana genome, representing ~50% annotated gene ontology (GO) functional categories. Approximately 14% of genes with GO annotations corresponding to regulation of gene expression were found to be under sRNA control. The unbiased bioinformatic approach used to produce the network was able to detect 107 PHAS loci (regions of phasiRNA production), 5,047 active phasiRNAs (~70% of which were non-canonical), and reconstruct 17 regulatory modules resulting from complex regulatory interactions between different sRNA-regulatory pathways. Known regulatory modules like miR173-TAS-PPR/TPR and miR390-TAS3-ARF/F-box were faithfully reconstructed and expanded, illustrating the accuracy and sensitivity of the methods and providing confidence for the validity of findings of previously unrecognized modules. The network presented here includes a 2X increase in the number of identified PHAS loci, a large complement (~70%) of non-canonical phasiRNAs, and the most comprehensive evaluation of sRNA cleavage activity in A. thaliana to date. Structural analysis showed similarities to networks of other biological systems and demonstrated connectivity between phasiRNA regulatory modules with extensive co-regulation of transcripts by miRNAs and phasiRNAs. The described regulatory network provides a reference that will facilitate global analyses of individual plant regulatory programs such as those that control homeostasis, development, and responses to biotic and abiotic environmental changes.