Total Nucleic Acid Research Articles

Analysis of microbial composition and sharing in low-biomass human milk samples: a comparison of DNA isolation and sequencing techniques

Human milk microbiome studies are currently hindered by low milk bacterial/human cell ratios and often rely on 16S rRNA gene sequencing, which limits downstream analyses. Here, we aimed to find a method to study milk bacteria and assess bacterial sharing between maternal and infant microbiota. We tested four DNA isolation methods, two bacterial enrichment methods and three sequencing methods on mock communities, milk samples and negative controls. Of the four DNA isolation kits, the DNeasy PowerSoil Pro (PS) and MagMAX Total Nucleic Acid Isolation (MX) kits provided consistent 16S rRNA gene sequencing results with low contamination. Neither enrichment method substantially decreased the human metagenomic sequencing read-depth. Long-read 16S-ITS-23S rRNA gene sequencing biased the mock community composition but provided consistent results for milk samples, with little contamination. In contrast to 16S rRNA gene sequencing, 16S-ITS-23S rRNA gene sequencing of milk, infant oral, infant faecal and maternal faecal DNA from 14 mother-infant pairs provided sufficient resolution to detect significantly more frequent sharing of bacteria between related pairs compared to unrelated pairs. In conclusion, PS or MX kit-DNA isolation followed by 16S rRNA gene sequencing reliably characterises human milk microbiota, and 16S-ITS-23S rRNA gene sequencing enables studies of bacterial transmission in low-biomass samples.

Analytical and Clinical Validation of Duoseq, a Novel Assay for Rapid, on-Site Clinical DNA and RNA Sequencing of Hematologic Malignancies

Introduction: Next generation sequencing (NGS) has become a critical component of the workup of malignancies. NGS can provide important diagnostic information including mutations, chromosomal copy number alterations and translocations (from DNAseq) as well as gene expression and fusions (from RNAseq). Incorporation of NGS in the clinic remains difficult. DNAseq and RNAseq remain distinct assays with challenging bioinformatics and turnaround times (TAT) that can last several weeks. Duoseq is a novel assay for combined DNA- and RNAseq in a single workflow that detects single nucleotide variants (SNVs), insertions/deletions (indels), splice site variants (SSVs), structural variants (SVs) and Epstein-Barr virus (EBV) status. Initially implemented for hematologic malignancies, Duoseq assesses nearly all known gene drivers (N=475) and is compatible with both fresh-frozen and formalin-fixed, paraffin-embedded (FFPE) tissues. It is available as a reagent kit and includes cloud-based bioinformatics, enabling any clinical lab with an Illumina sequencer to perform NGS in-house with a TAT of 2-3 days. Methods/Results: Duoseq was validated for concordance with commercial DNA- and RNAseq kits (New England BioLabs (NEB)). Sequencing libraries were prepared on DNA alone, RNA alone and total nucleic acid (TNA) from cell lines in accordance with kit protocols and captured using the Duoseq panel. Results were equivalent between Duoseq and individual (NEB) library preparations with nearly complete concordance in DNA variants (99.4%, P<10 -6) and RNA expression (R 2=0.97, P<10 -6). Separately, copy number alteration calls across 42 cases had high concordance between Duoseq and Affymetrix array approaches (P<10 -6). Analytical validation was performed at two CLIA laboratories to establish assay precision and reproducibility, sensitivity (LoD), and specificity. Analytical validation was performed on well characterized and control samples that were selected to harbor an array of clinically important variants at known allele frequencies. Precision and reproducibility testing confirmed Duoseq's ability to produce accurate results independent of external variables. We tested inter-lab reproducibility, intra-day and inter-day repeatability and technician-to-technician reproducibility. In each case, Duoseq achieved >98% precision and reproducibility for each variant class (SNVs, indels/SSVs and SVs). We confirmed the LoD using TNA from the same samples diluted in normal TNA to create 3-point curves that spanned our expected LoD. We confirmed the assay to be 100% accurate down to ≥5% mutant allele fraction (MAF) for SNVs, ≥10% MAF for indels/SSVs, and ≥20% tumor purity for SVs. Specificity was additionally assessed by including normal cell lines in each run to determine the incidence of cross-contamination indicated by false positives. Analytical specificity was 100%, indicating no instances of sample cross-contamination. We assessed the clinical accuracy of Duoseq using FFPE myeloid and lymphoid specimens (N=153) collected at both laboratories and characterized via orthogonal methods including Foundation Medicine (SNVs and indels/SSVs), fluorescent in situ hybridization (translocations) and in situ hybridization (EBV status). Duoseq achieved a positive predictive agreement of 98% for SNVs (N=139), 96% for indels/SSVs (N=55), 95% for translocations (N=282), and 100% for EBV status (N=58). Cell of origin (COO) for diffuse large B cell lymphoma (n=42) using Hans criteria and had high concordance (90%) with the COO calls rendered from Duoseq expression data. Given Duoseq's array of measurements and short TAT, we plan a potential application in “match” clinical trials where patients are enrolled in treatment arms based on molecular profiling. In ongoing work to test a potential trial workflow, we enrolled four institutions that will provide cases to a central site for pathology review and molecular profiling enabling trial arm assignment. Specimen enrollment is anticipated to be completed to allow for results to be presented at the meeting. Conclusions: Duoseq is comparable to current methods and provides a cost and time effective option for library preparation.The large gene panel enables testing a broad range of clinical features in a single assay, including genes required for diagnosis and prognostication in heme malignancies. These results support clinical use of Duoseq.

RNA Fusions and Their Association with DNA Alterations in Myeloid Neoplasia Patients Identified By a Single Tube Multimodal Comprehensive Genomic Profiling Test

Background: Recent updates into NCCN professional guidelines have included adding several genomic biomarkers for myeloid disorders. Detecting SNVs, indels, select CNVs and genomic rearrangements in a single comprehensive genomic profiling test is invaluable for clinical care. Moreover, WHO recognizes 23 genomic rearrangements or fusions which define subclasses of AML, MDS/MPN and related neoplasms, and their detection is essential for patient management. Here we present joint prevalence data of gene fusions and other genomic alterations in myeloid disorders in a cohort of 312 patients analyzed by a CLIA grade single-tube NGS assay capable of concurrent analysis of DNA and RNA alterations. Methods: Total nucleic acid (TNA) from bone marrow or peripheral blood was analyzed by a CLIA grade custom amplicon-based multimodal NGS test reporting DNA mutations in 126 genes and gains/losses in 17 genes by DNA-seq, and RNA fusions from 40 genes by RNA-seq. Libraries were sequenced on a NovaSeq6000 instrument, and fusions were called with in-house developed BI pipeline using the distribution of AI-assisted fusion confidence scores to improve the signal to noise discrimination for fusion calls before validation. Deidentified patient data was used according to an IRB approved protocol. Results: Analytical validation of RNA fusion calling against FISH and Sanger-seq in 74 hematologic disorder samples demonstrated 96.7% sensitivity and 98.2% specificity with 100% reproducibility. This improved fusion detection module was added to our CLIA validated NGS assay, which at tumor purity of >20%, already detects SNVs (VAF >3%), Indels (<81 bp, VAF >3%) and copy number gains/losses with a sensitivity of 98.3%, 98.9 % and 96 % for SNV, Indels and copy number gains/losses, respectively and a specificity of >99.9% for SNV/Indels and 98.4% for gene gains/losses. Next, data from a total of 789 patients serially tested with this assay was used to study the distribution of myeloid fusion events in community cases which included 312 adult patients with confirmed/suspected myeloid disorders including AML, CML, MDS/MPN and other disorders while 477 patients had a diagnosis of lymphoid leukemias. Fifty five (55) % of the Myeloid leukemia patients were male and 45% female, with a median age of 67.5 (22-87) and 71 (22-89) years, respectively. 27% (84/312) of them presented a gene fusion, of which in 21% of cases it involved a gene from WHO/NCCN fusion gene recommendations. Prevalence for common fusions were 6.7% for BCR::ABL1; 5.1% for PML::RARA; 2.6% (8/312) for KMT2A; 1.9% for RUNX1::RUNX1T1; 1.6% CBFB::MYH11; 1.3% for PICALM::MLLT10 and 1 % for NUP98. Other fusions were detected in <1% patients. Fusions of PDGFRA, ETV6, ZNF384, FGFR1 and other genes were also observed. BCR::ABL1 fusions were seen not only in CML patients but also in a patient with AML. For KMT2A, 25% (2/8) of fusions detected by NGS in all patients were confirmed by Sanger-seq but missed by FISH, which correlates with higher sensitivity of the NGS assay. Interestingly, the prevalence of fusion positive cases on the lymphoid leukemia patients (17%, 80/477) tested on the same period of time. Novel fusions were called in ~8% of all patients with high confidence. We validated by Sanger sequencing the CCND2::MGP fusion, which was identified on a patient with AML. This fusion protein is predicted to be oncogenic as it lacks the region from CCND2 including Thr280, a residue required for ccnd2 degradation. Finally, we analyzed the relationship between fusions and recurrently mutated genes. We found that DNA alterations in CALR, EZH2, FLT3, KIT, and ZRSR2 were enriched in fusion positive cases. Conversely alterations on a subset of genes were absent on fusion positive cases and enriched on fusion negative cases. Those included alterations on CEBPA, IDH1/2, KMT2A, MPL, NPM1, BCOR, IKZF1, FBXW7 STAG2, CSF3R, PDGFRA, PHF6, PTPN11. Conclusions: A robust low-noise RNA fusion detection assay coupled us DNA alterations on myeloid disorders in a single assay enables to fully molecularly characterize acute myeloid leukemias and other myeloid disorders. Frequencies of well-known fusions in a small community-based cohort were similar to studies performed in academic settings with subsets of gene alterations being mutually exclusive from fusions. Larger studies are needed to confirm those associations.

Diagnosis of Peach latent mosaic viroid (PLMVd) and Hop stunt viroid (HSVd) by RT-PCR using different extraction protocols

RT-PCR method was performed using four different nucleic acid extraction methods to identify viroids; peach latent mosaic viroid (PLMVd) and hop stunt viroid (HSVd) which causes serious damage to stone fruits. Leaf samples were collected from fruit orchards showing viroid-like symptoms in İzmir province. Silica capture, citric buffer, lithium chloride, and ames buffer methods were used to extract total nucleic acids. The four extraction methods were compared using samples collected during the vegetation period from naturally infected trees (plum, apricot, and peach). They were evaluated with RT-PCR tests. In 64 stone fruit tree samples, only the silica capture method gave reliable and accurate results in RT-PCR molecular tests for the detection of PLMVd and HSVd. The other wielded nucleic acid extraction methods were found to be ineffective for the isolation of the viroid RNAs.

Comparison of metagenomic and targeted methods for sequencing human pathogenic viruses from wastewater.

Most public health initiatives that monitor viruses in wastewater have utilized quantitative polymerase chain reaction (PCR) and whole genome PCR sequencing, mirroring techniques used for viral epidemiology in individuals. These techniques require prior knowledge of the target viral genome and are limited to monitoring individual or small groups of viruses. Metagenomic sequencing may offer an alternative strategy for monitoring a broad spectrum of viruses in wastewater, including novel and emerging pathogens. In this study, while amplicon sequencing gave high viral genome coverage, untargeted shotgun sequencing of total nucleic acid samples was unable to detect human pathogenic viruses with enough sensitivity for use in genomic epidemiology. Enrichment of shotgun libraries for respiratory viruses using hybrid-capture technology provided genotypic information on a range of viruses simultaneously, indicating strong potential for wastewater surveillance. This type of targeted metagenomics could be used for monitoring diverse targets, such as pathogens or antimicrobial resistance genes, in environmental samples.

A Simplified, Efficient and Rapid DNA Extraction Protocol from Rice Grains and Leaf

DNA extraction is very complicated with different plant species because the presence of secondary metabolites that hinder with DNA isolation processes and application like DNA restriction, Gene amplification, as well as gene cloning. A simple method for preparation of rice genomic DNA was developed. The current study expressed comparatively rapid, cost-effective, less time consuming, methods for DNA extraction from seed, young leaves and old leaves of rice genotypes without using liquid nitrogen, phenol, and β–mercaptoethanol in extraction buffer. Using this method, high quality and quantity of genomic DNA was get from 0.5 g of rice seed, young leaves and 75 days old leavesand extracted total nucleic acids (genomic DNA) were amplified by employing single sequence repeats (SSR) or microsatellite markers, which produced reproducible results.This protocol resolves the problems of DNA degradation, contamination, and low yield due to binding and/or coprecipitation with starches and polysaccharides.

Border disease virus and chlamydophila abortus co-infection in aborted sheep foetuses

Abortion in sheep and goats is one of the most important reproductive problems that affect small ruminants breeding. Non-infectious factors and infectious agents such as bacterial, viral, mycotic and parasitic infectious agents can cause abortion in sheep and goats. A high rate of abortion (43.75%) was observed in two sheep flocks in Niğde Province in Turkey during the lambing season in 2016. To determine the reason of abortion, aborted sheep foetuses (n = 3) and EDTA whole blood samples (n = 3) from mother of the foetuses were collected from two sheep flocks. Aborted foetuses and buffy coat cells of the EDTA whole blood samples were used for total nucleic acid extraction. Extracted nucleic acids were analysed for akabane virus (AKAV), bluetongue virus (BTV), peste des petits ruminants virus (PPRV), pestiviruses, Schmallenberg virus (SBV), Brucella spp., Chlamydophila abortus (C. abortus), Coxiella burnetii and Listeria monocytogenes. Border disease virus (BDV) RNA and C. abortus DNA were detected in three aborted sheep foetuses whereas other investigated infectious agents were not detected. Additionally, EDTA whole blood samples from mother of the foetuses were also found BDV positive. To the best of my knowledge, this is the first report on BDV and C. abortus co-infection in aborted sheep foetuses.

Osimertinib as a Potential Targeted Therapy for Non-Small Cell Lung Carcinoma (NSCLC) Patients with EGFR Exon 20 T790M

BACKGROUND: Emergence of drug resistance due to epidermal growth factor receptor (EGFR) Exon 20 T790M poses a challenge in the effective management of non-small cell lung carcinoma (NSCLC). Significant breakthrough in the management of NSCLC with a specific genetic alteration causes substantial condition improvement in patients whose cancer progressed after first-generation tyrosine kinase inhibitor treatment and who developed tumors with EGFR Exon 20 T790M mutation. The present study analyzed a cohort of NSCLC patients and investigated the incidence of the EGFR Exon 20 T790M status with Osimertinib therapy, along with its impact on survival rates.METHODS: This was a retrospective cohort study on 22 NSCLC subjects who were genetically examined for EGFR status from plasmic cell free total nucleic acid. Subjects with EGFR Exon 20 T790M mutation were treated with/without Osimertinib. Demographic and clinical data were descriptively summarized, and the differences of each variable and correlation between survival rate and EGFR Exon 20 T790M were analyzed.RESULTS: Subjects with (n=13) and without (n=9) EGFR Exon 20 T790M had survival rates of 10.77±2.45 and 4.78±1.48, respectively (p=0.000). Based on 1-year survival status of subjects with EGFR Exon 20 T790M, there were 3 Osimertinib-treated survivors and 2 Osimertinib-treated non-survivors. Eight subjects with EGFR Exon 20 T790M and without Osimertinib treatment did not survive (p=0.001).CONCLUSION: Since the treatment of Osimertinib demonstrated a noteworthy survival rate among NSCLC subjects with EGFR Exon 20 T790M, thus Osimertinib could be suggested as a potential targeted therapy for NSCLC subjects with EGFR Exon 20 T790M.KEYWORDS: non-small cell lung carcinoma, EGFR, Exon 20, T790M, osimertinib

Respiratory virus surveillance in hospitalized children less than two-years of age in Kenema, Sierra Leone during the COVID-19 pandemic (October 2020- October 2021).

Globally, viral pathogens are the leading cause of acute respiratory infection in children under-five years. We aim to describe the epidemiology of viral respiratory pathogens in hospitalized children under-two years of age in Eastern Province of Sierra Leone, during the second year of the SARS-CoV-2 pandemic. We conducted a prospective study of children hospitalized with respiratory symptoms between October 2020 and October 2021. We collected demographic and clinical characteristics and calculated each participant´s respiratory symptom severity. Nose and throat swabs were collected at enrollment. Total nucleic acid was purified and tested for multiple respiratory viruses. Statistical analysis was performed using R version 4.2.0 software. 502 children less than two-years of age were enrolled. 376 (74.9%) had at least one respiratory virus detected. The most common viruses isolated were HRV/EV (28.2%), RSV (19.5%) and PIV (13.1%). Influenza and SARS-CoV-2 were identified in only 9.2% and 3.9% of children, respectively. Viral co-detection was common. Human metapneumovirus and RSV had more than two-fold higher odds of requiring O2 therapy while hospitalized. Viral pathogen prevalence was high (74.9%) in our study population. Despite this, 100% of children received antibiotics, underscoring a need to expand laboratory diagnostic capacity and to revisit clinical guidelines implementation in these children. Continuous surveillance and serologic studies among more diverse age groups, with greater geographic breadth, are needed in Sierra Leone to better characterize the long-term impact of COVID-19 on respiratory virus prevalence and to better characterize the seasonality of respiratory viruses in Sierra Leone.

First report of Fig virus B in Ficus carica in New Zealand.

Fig (Ficus carica) has been cultivated since ancient times, and is now grown worldwide, both for its fruit and as an ornamental plant. Several viruses and viroids are associated with Fig mosaic disease (FMD), a disease complex occurring worldwide (Preising et al. 2021). Fig mosaic virus (FMV), fig leaf mottle-associated virus 1 (FLMaV-1), fig mild mottle-associated virus (FMMaV), and fig badnavirus 1 (FBV-1) are known to infect fig in New Zealand (Minafra et al. 2012; Veerakone et al. 2015). In December 2020, leaf samples from a fig tree growing on the roadside at St Heliers, Auckland, showing dieback with foliar chlorotic mosaic symptoms, was received for virus testing. Total nucleic acid was extracted from the symptomatic leaves using a KingFisher™ mL Purification System (Thermofisher Scientific, Waltham, MA) with an InviMag Plant DNA Mini Kit (Invitek Molecular GmbH, Germany) and subjected to high-throughput sequencing on an Oxford Nanopore Technologies MinION device using the method described in Liefting et al. 2021. All sequence analysis was performed using Geneious Prime 2021.1.1 (https://www.geneious.com). A total of 355,858 reads that passed quality check were subjected to BLASTn search against the NCBI nt database as described in Liefting et al. 2021. The following viruses produced hits: FMV, FBV-1, FMMaV and a fig closterovirus. The presence of FMV, FBV-1 and FMMaV were confirmed by species specific RT-PCRs. To identify the closterovirus, reads were mapped to closteroviruses reported in fig including the recently identified tentative species fig virus A (FiVA; GenBank accession no MN817232) and fig virus B (FiVB; GenBank accession no. MN817233). Five viral contigs ranging from 939 to 2,340 nucleotides (nt) were obtained from mapping to FiVB. Subsequently, a 6.4 kb sequence (GenBank accession no. OQ968551) from the 3' region of the NZ isolate was amplified by overlapping RT-PCR using primers designed from the contig sequences. The sequence shared 79.5% nucleotide (nt) identity with FiVB The original sample and a further 25 symptomatic and 10 asymptomatic fig samples, collected from the Auckland area between 2016 and 2021, were tested using FiVB specific RT-PCR and Sanger sequencing using primers FiVB-F1 (5'-GAGGGAGAGATGTAGATGC-3') and FiVB-R2 (5'-TGTCGTCGATATCGTTGTGT-3'), designed to amplify a 725 nt fragment in the 70 kDa heat shock protein (HSP70) ORF. Products of the expected size were amplified from the original sample and three symptomatic samples and their sequences found to be identical. BLAST searches showed that the sequence (GenBank accession no. ON553403) shared 82.7% nt and 87.3% amino acid (aa) identity to an isolate of FiVB (GenBank accession no MN817233). These additional positive samples were collected from a small home nursery where the plants were propagated from cuttings and have been distributed locally, suggesting the virus is very likely to have a limited spread throughout the Auckland area. All three FiVB infected samples were also positive for FMV. However, the association of FiVB with FMD symptoms is unknown. FiVB was first identified from a latex sample exuded from a fig tree collected from Japan (Park et al. 2021) and is the only report of FiVB in the world to date. Although an identical sequence from Argentina, named fig closterovirus 1, was submitted to GenBank, the origin of this isolate is not known. To our knowledge, this is the first report of FiVB in New Zealand.

Non-Small Cell Lung Cancer: Targetable Variants in Concurrent Tissue and Liquid Biopsy Testing in a North Indian Cohort.

Testing for EGFR, ALK, ROS1 and MET alterations in paired tissue and plasma samples of treatment-naïve patients of NSCLC and correlating their status with overall survival. One hundred treatment-naïve patients were recruited after obtaining informed consent. Ten ml of blood was collected within a period of two weeks from histological diagnosis, prior to the start of any treatment. DNA & RNA extraction was done from formalin-fixed paraffin embedded (FFPE) tissue and total cell-free nucleic acid extraction was done from plasma samples. EGFR mutation, ALK, ROS1 and MET rearrangements were tested by ARMS (Amplification Refractory Mutation System) PCR. All statistical analyses were conducted in R version 4.1.1. A total of 61 cases showed molecular alterations in tissue samples which included EGFR mutations (47), ALK rearrangements (12), ROS1 fusion (2). MET alteration was not detected. Forty-three cases showed EGFR mutations in plasma, 26 of which were concurrently positive in tissue. Concordance observed was 62%. ALK-EML4 rearrangement, ROS1 fusion and MET were not detected in plasma samples. Sensitivity and specificity for detection of EGFR mutation in plasma were 55.3% and 67.9% respectively. Univariate Cox regression analysis showed a positive association between EGFR mutation in tissue and overall survival (HR = 0.4; 95% CI: 0.2-0.7; p = 0.003) and improved overall survival in those who received targeted therapy (HR = 0.29; 95% CI: 0.1-0.8; p = 0.02). Concurrent testing in tissue and liquid biopsy in NSCLC increased the detection of EGFR mutations (47% to 64%). This has substantial implications in deciding treatment and administration targeted therapy and the consequent overall survival.

A-269 Post-pandemic Opportunities for Commercial Laboratories in Infectious Disease Diagnostics and Public Health

Abstract Background Aegis launched COVID-19 testing with a daily capacity of 3000 tests and scaled to over 100 000 tests/day in one year. The lab has performed over 14.2M COVID-19 tests to date, servicing a broad network of physicians, urgent care centers, nursing homes, public schools, departments of health, and retail pharmacies across the United States. As SARS-CoV-2 infections reach an equilibrium, Aegis recognized the opportunity to address broader public health needs and leveraged the partnerships, technologies, and infrastructure developed to build the Infectious Disease Testing (IDT) menu aimed at limiting the spread of health risks within the community. Methods Real-time polymerase chain reaction (RT-PCR) tests for common and rare bacterial, viral, and parasitic pathogens associated with genital health (GH) and sexually transmitted infections (STI), as well as infections of the urinary (UTI), gastrointestinal (GI), and respiratory tracts (RTI) were validated. These tests are performed in 384-well custom PCR plates, pre-spotted with primers and probes capable of detecting the presence of DNA or RNA (LOD = 10 copies/µL) from 30 different organisms in up to 12 patient samples simultaneously. Syndromic panels were developed for ordering based on similar intended use, and providers have the option to order panels or stand-alone tests based on medical necessity. Following receipt into the lab, the total nucleic acid is extracted from the primary specimen and pipetted into the PCR assay plate with Taq DNA polymerase master mix, and analysis and reporting of ordered targets are complete within nine hours, on average. Results and de-identified demographic data are parsed from the laboratory information management system (LIMS) to Microsoft PowerBI software to internally track testing trends, positivity rates, and outbreaks of infectious diseases. Results Historical data including ordered profiles, matrices, and pathogens detected combined with de-identified demographic data such as patient age, gender, race, residing state, and collection month were imported into PowerBI and used to create interactive visuals for a dashboard to monitor trends and predict future testing patterns. The IDT panels ordered most frequently include STI (27.1%), upper RTI (23.6%), bacterial vaginosis (21.0%), GI bacterial (15.1%), candidiasis (13.7%), and GI viral (13.0%). The majority (74%) of GH/STI tests were ordered for patients between the ages of 15 to 34 years, and the most prevalent infections detected were bacterial vaginosis (35%), mycoplasma hominis (22%), and chlamydia trachomatis (4%). Sixty-one percent (61%) of upper RTI tests were ordered for patients between the ages of 0 to 14 years, and fluctuations in positivity rates followed seasonal trends for pathogens such as rhinovirus, influenza, adenovirus, RSV, and enterovirus D68. GI profiles were predominantly ordered for the younger (0–14 years, 35%) and the older (>65 years, 18%) population with e. coli O157 (11%), norovirus (9%), sapovirus (6%), and astrovirus (4%) infections detected at the highest rates. Conclusion The COVID-19 pandemic underscored the importance of molecular diagnostics for SARS-CoV-2 infection and surveillance. Experience gained during the COVID-19 response allowed Aegis to repurpose the resources, capacity, and infrastructure to create business development opportunities aimed at laboratory growth in infectious diseases and public health.

B-257 Rapid Implementation of Monkeypox Detection in a Brazilian Diagnostic Laboratory

Abstract Background Monkeypox is a double-stranded DNA virus (approximately 197 kbp) belonging to the genus Orthopoxivirus. It is responsible for sporadic outbreaks in countries in Africa. Recently, in May 2022 a patient with a recent travel history to Nigeria tested positive for monkeypox in the UK. After that, people with symptoms (usually epithelial pustular lesions) were diagnosed with the virus in several countries, such as Portugal and the United States. In Brazil, the first case was confirmed in June 2022, the patient had traveled to Spain and Portugal. It was in this context that the Technical Operational Nucleus of our laboratory, Sabin Diagnosis and Health, located in Brasilia, capital of Brazil, validated and implemented a test to detect the genetic material of Monkeypox. Thus, in this work we present the implementation of this test. Method For the detection of Monkeypox viral DNA we chose to implement a real-time PCR (qPCR) methodology. The validated samples were swabs and wound scabs. The nucleic acid extraction chosen was using the Virus Pathogen kit from (QIAGEN), ‘midi’ protocol in the qiasymphony equipment (QIAGEN). Later we validated the extraction with the Maxwell RSC Viral Total Nucleic Acid Purification Kit extraction kit (Promega). For qPCR, we selected monkeypox-specific primers and probes, which were not able to recognize other Orthopoxiviruses (such as human pox) to ensure specificity. We used RNASEP-specific primers as an endogenous extraction control. As a positive control (PC) we used a synthetic double-stranded DNA fragment with the target sequence flanked by 10 bp. The limit of detection of the test was performed using serial dilutions of the positive control. Furthermore, given the initial absence of a positive sample for validation, we designed four pairs of primers that amplified four different regions of the viral genome for sequencing by the Sanger technique. The first positive samples were confirmed by sequencing these four regions. Results The protocol was designed in the first week after the confirmation of the first case in Brazil. The first tests with the PC showed the effectiveness of the primers. After adjusting the reaction conditions to assess the specificity of the test, we tested the protocol with samples known to be positive for other viral pathogens and obtained negative results for all of them, confirming the high specificity. After that we experimentally determined the limit of detection (25 copies per reaction). On July 8, 2022, one month after the first confirmed case in Brazil in the state of São Paulo, we released the Monkeypox detection test with the methodology described above. Since then, we have performed 193 tests, of which 59 (31%) were positive, most of them (56–95%) in men. The first 10 positive results were confirmed by genetic sequencing (Sanger). Discussion The implementation of the Monkeypox detection test by our laboratory allowed a quick response for Brazilian patients who needed the test after the virus entered the country.

Analysis of Coconut cadang-cadang viroid variants on field samples exhibiting variation in orange spotting symptom expression and severity.

Sequence variation has been attributed to symptom variations but has not been investigated in Orange Spotting-Coconut cadang-cadang viroid (OS-CCCVd) infected palms. Likewise, the relationship between Coconut cadang-cadang viroid (CCCVd) variants, Orange Spotting (OS) severity and the accumulation of the viroid in the palms have not been elucidated. This paper describes the characterization of CCCVd variants by cloning and sequencing, followed by correlation with symptom expression. Total nucleic acids were extracted from leaf samples harvested from frond 20 of seven Dura×Pisifera (D×P) African oil palm (Elaeis guineensis Jacq.) aged between 13 and 21years old collected from local plantations. The nucleic acids were fractionated using 5% non-denaturing polyacrylamide gel electrophoresis (PAGE) before being subjected to detection by reverse transcribed polymerase chain reaction (RT-PCR). The PCR products were cloned into a plasmid vector and the sequence of the clones was analyzed. CCCVd variants were quantified using real-time qPCR assay with CCCVd specific primers. Sixteen randomly selected clones of (OP246) had an arbitrary 100% identity with CCCVdOP246 (GeneBank Accession No: HQ608513). Meanwhile, four clones had >93% similarity with several minor sequence variations forming variants of OP234, OP235, OP251 and OP279. The OS symptoms observed in the field were characterized into three categories based on the size and morphology of the orange spots on the affected fronds. In addition, there was no direct correlation between disease severity and the accumulation of CCCVd variants in oil palm. This finding is the first report describing the sequence variation of the CCCVd RNA and symptom variation in OS oil palm field samples.

Agave attenuata and Agave amica, new hosts of Tuberose mild mosaic virus in Mexico.

Agave attenuata is a Mexican wild plant originally from highlands in the central and occidental mountains of Mexico. This species, known as "swan´s neck agave", is used only as an ornamental plant in public and private gardens. No virus had previously been reported from A. attenuata before this study. In a survey conducted in a commercial greenhouse in Cuautla, Morelos, in 2018, several plants were observed with symptoms of green mosaic and streaks, consistent with a putative viral infection. Sap inoculation from symptomatic A. attenuata plants to herbaceous indicator plants (Nicotiana benthamiana and N. tabacum) failed to produce symptoms in the mechanically inoculated plants. ELISA specific test to CMV, TEV, AMV, TMV and Potyvirus Group (Agdia, Inc.), was positive only for the last one (Chen and Chang, 1998). To determine the identity of the potyvirus involved, total nucleic acid extracts from 100 mg of symptomatic leaves (Trizol reagent; Gibco BRL Life Technologies, England) were used as template in RT-PCR with genus-specific potyvirus primers POT1-POT2, which targeted the variable 5´ terminal half of the coat protein gene of potyviruses (Colinet et al. 1998). The expected 900 bp amplicon was consistently detected in 10 symptomatic A. attenuata plants whereas no PCR products were obtained from 15 asymptomatic A. attenuata plants collected from the "Agaves de México" section at the 'Botanic Garden' of the Instituto de Biología de la UNAM, México. The amplicons were sequenced by the Sanger´s method and the obtained nucleotide (nt) sequences (Acc. No KY190217.1; OP964597-598) and their derived amino acid (aa) sequences were 94.68% to 95.80% similar to an isolate of Tuberose mild mosaic virus (TuMMV; Potyvirus; (Acc. No ON116187.1) characterized from Agave amica in India (Raj et al. 2009). Interestingly, A. amica (formerly Poliantes tuberose) is also a wild Mexican plant that is geographically distributed in the central and south regions of Mexico and is currently being commercially cultivated as an ornamental plant. Plants of A. amica (n=10) showing yellow mild streak were collected from commercial greenhouse and tested positive for TuMMV by RT-PCR and Sanger sequencing (No Acc. OP964599-601 levels) described above. The derived TuMMV sequences from A. attenuata and A. amica were 99-100% similar to each other at the nt/aa level. To exclude the involvement of additional viral agents in the disease, high-throughput sequencing analysis was performed separately for each species of Agave on total RNA extracts from a composite sample of symptomatic leaf tissues using Illumina´s Next Seq 500 platform. Analysis of the obtained 13,260,700 reads (each 75 nt) by the Trinity software, with a total number of sequences of 22,793, resulted in the identification of a single viral contig of 9500 nt for A. attenuata (Acc. No OP964595). Similarly, for A. amica, 27,262,248 reads were obtained, with a total number of sequences of 23,269, resulting in the identification of a single viral contig of 8500 nt (ACC. No OP964602). These contigs showed an identity percentage of 96%/88% and 98%/96% for nucleotides and amino acids, respectively, compared to an isolate of TuMMV from India (Acc. OM293939). Mexico is a center of origin for numerous species of genus Agave which have high economic, social, and ecological impact. TuMMV could be a threat to these plants and potentially to other unknown susceptible crops. To our knowledge, this is the first report of TuMMV in A. attenuata and A. amica in Mexico. REFERENCE Chen, C. C., and Chang, C. A. 1998. Characterization of a potyvirus causing mild mosaic on tuberose. Plant Dis. 82:45-49. Colinet, D., Nguyen, M., Kummert, J., Lepoivre, P., and Xia, F. Z. 1998. Differentiation among potyviruses infecting sweet potato based on genus- and virus-specific reverse transcription polymerase chain reaction. Plant Dis. 82:223-229. Raj, S.K., Snehi, S.K., Kumar, S., Ram, T. and Goel, A.K. 2009. First report of Tuberose mild mosaic potyvirus from tuberose (Polianthes tuberosa L.) in India. Australasian Plant Dis. Notes 4, 93-95.

Seasonality of Amblyomma americanum (Acari: Ixodidae) Activity and Prevalence of Infection with Tick-Borne Disease Agents in North Central Oklahoma.

Background: Amblyomma americanum is the most common tick infesting both animals and humans in the southern United States and transmits a variety of zoonotic agents. The rise in tick-borne diseases (TBD) globally imparts a need for more active surveillance of tick populations to accurately quantify prevalence and risk of tick-borne infectious organisms. To better understand TBD risk in north central Oklahoma, this study aimed to describe the current seasonal activity of A. americanum in this region and investigate the seasonality of tick-borne infectious agents. Materials and Methods: Tick collections were performed twice a month for a duration of 2 years at a field site in Payne County, Oklahoma. Total nucleic acid was extracted from a subset of adult A. americanum and tested for Rickettsia spp., Ehrlichia spp., and Borrelia spp. using established PCR protocols. Results: Peak activity times for each life stage were observed, with adults primarily active 1 month earlier than historical seasonal trends describe, and male A. americanum active earlier in the year than female A. americanum. Rickettsia spp., Ehrlichia chaffeensis, Ehrlichia ewingii, and Borrelia lonestari were found in 26.4%, 6.1%, 2.5%, and 1.1% of adult A. americanum, respectively. No seasonal trend in spotted fever group Rickettsia spp. (SFGR) was observed in peak activity months. Conclusions: This study found an apparently shifting phenology for A. americanum adults in Oklahoma. While these results did not show a trend in SFGR, further investigation is needed to better understand the potential seasonality of infection prevalence within A. americanum across the expanding range of this vector, especially considering the extended activity of males in winter months.

Hypoxia-associated genetic signature in ovarian steroid cell tumor NOS.

Steroid cell tumors, not otherwise specified (SCT-NOS), are uncommon ovarian neoplasms accompanied by virilization symptoms due to hyperandrogenism, which are malignant in approximately one-third of the cases. Given the rarity of SCT-NOS, their molecular underpinnings have not yet been studied in depth. In this case series, we performed the first comprehensive analysis of the genetic landscape of this rare ovarian tumor. A detailed clinicopathological description of an index case is also provided. Over a 20-year period, a total of eight patients were seen at our institution. Total nucleic acids (RNA and DNA) were extracted from evaluable formalin-fixed, paraffin-embedded tumor specimens (n = 7) and subjected to TruSight Oncology 500 testing and/or exome sequencing. The results identified pathogenic variants in several hypoxia-related genes - including HIF1A, VHL, SDHB, SRC, IDH2, and FOXO4. As the first comprehensive genetic analysis of SCT-NOS, this study shows that dysregulation in the hypoxia signaling pathway is a key molecular feature of this rare tumor. Clinically, long-term follow-up with periodic measurements of androgen levels should be pursued in all cases since recurrences may occur several years after the initial diagnosis.

First report of tobacco rattle virus infecting Weigela florida in the USA.

Weigela (Weigela florida (Bunge) A. DC., Family: Caprifoliaceae) are woody shrubs native to North China, Korea, and Japan. In the U.S., weigela are commonly used as landscape ornamental plants (McNamara et al. 2010). Two viruses have been reported in weigela: tomato spotted wilt orthotospovirus and impatiens necrotic spot orthotospovirus (Sastry et al. 2019). Ten weigela plants, originating from commercial nurseries in Minnesota, exhibiting chlorosis, chlorotic line patterns, and necrosis (e-Xtra) were submitted for virus diagnostics as potted plants at the University of Minnesota Plant Disease Clinic and the Virology Lab in 2019 and 2020 (five plants each year). Under greenhouse conditions, symptoms progressed from chlorosis to necrosis and even plant death in two of the five plants in 2019. Electron microscopy revealed rod-shaped particles of ≈20 nm in diameter and lengths between 40-200 nm with similar morphology to members of the genus Tobravirus (e-Xtra). Virus-like particles were enriched by ultracentrifugation and total nucleic acids were extracted from partial purifications using a phenol:chloroform extraction method (Lockhart et al. 1997). Tobacco rattle virus (TRV) was identified by cloning and sequencing of the 463bp amplicon obtained with the TRV detection primers described in Robinson, 1992. High-throughput sequencing (HTS) was done to confirm the TRV detection. A cDNA library was prepared from purified viral RNA using the TruSeq Stranded mRNA kit and sequenced on Illumina NovaSeq 6000 platform as 150 bp-paired end reads. A total of 44,316,446 raw data reads were obtained, preprocessed using the BBDuk plugin, and de novo assembled using SPAdes assembler. Viral contigs were identified using the NCBI BLASTX tool. The assembly of TRV RNA1 was 6,842 nt with 20,627,348 reads (47% of total reads) mapped to it and an average coverage per nucleotide at 323,639X. The assembly of TRV RNA2 was 3,033 nt with 22,769,253 reads (52% of total reads) mapped to it and an average coverage per nucleotide at 798,660X. NCBI GenBank accession numbers for the assemblies representing RNA1 and RNA2 are OQ408335 and OQ408336, respectively. NCBI BLASTn analysis showed the highest level of nucleotide identity to TRV genomic RNA segments 1 and 2, with 97% and 99% identity to the TRV isolate RNA1 (GQ903771) and RNA2 (GQ903772), respectively, that originated from Michigan potato. No other viral contigs were detected from the virion nucleic acid extraction by HTS, however this enrichment method doesn't exclude other viruses. In addition to using the detection primers by Robinson 1992, we designed primers based on our HTS data: TRV-WG-DetF3 5'- GACGAAGGAGGCTGTCATTGC-3' and TRV-WG-DetR3 5'-CGGACTATCGTGATGCCCATGC- 3'. RT-PCR amplicons from each of the 10 symptomatic plants were cloned and sequenced. Among these clones, Sanger sequence identities ranged between 96-100% compared to the HTS data and 98-99% to the TRV potato isolate from MI. To our knowledge, this is the first report of TRV infecting the ornamental host W. florida worldwide. TRV is a nematode-transmitted viral pathogen of economic importance, most notably in potatoes (Sastry et al. 2019). In the US, TRV has been reported on several landscape ornamentals, horticultural crops, and native habitats. Further research is needed to investigate the impact of TRV on the ornamental industry and the role of ornamentals as reservoirs for cultivated crops like potatoes.

Geographical distribution of tomato-infecting begomoviruses in major cucurbits in India: a diagnostic analysis using begomovirus species specific PCR.

Cucurbits are an essential summer-season vegetable crops, but they are highly vulnerable from a range of abiotic and biotic factors. One of the significant biotic factors posing a growing menace to the production of major cucurbits in India is the emergence of tomato-infecting begomoviruses. In this study, we utilized PCR-based species-specific primers, developed earlier in our laboratory for the detection of begomoviruses infecting tomato and chilli plants, to identify begomoviruses in cucurbits across various regions of India. Leaf samples from major cucurbits were collected from different regions of Haryana, Delhi, Uttar Pradesh, Chhattisgarh, Maharashtra, Telangana and Karnataka, during the year 2020-2021. Total nucleic acid (TNA) was extracted from the samples and subjected to PCR using a generic primer specific to begomoviruses. The samples that exhibited positive amplification were further tested using six different species-specific primers targeting specific begomovirus species, namely Tomato leaf curl New Delhi virus (ToLCNDV), Tomato leaf curl Palampur virus (ToLCPalV), Tomato leaf curl Bangalore virus (ToLCBV), Tomato leaf curl Joydebpur virus (ToLCJoV), Tomato leaf curl Gujarat virus (ToLCGuV), and Chilli leaf curl virus (ChiLCV). The PCR analysis revealed that among the 551 plant samples tested, a total of 124 samples exhibited positive amplification using the universal begomovirus PCR. Specifically, 47 samples tested positive for ToLCNDV, 73 samples were positive for ToLCPalV and only one sample showed positive amplification for ChiLCV. However, none of the samples tested positive for ToLCJoV, ToLCGuV and ToLCBV. These findings from our study indicate the prevalence of ToLCNDV and ToLCPalV in major cucurbits across India. Furthermore, the study highlights the varied distribution of begomoviruses in major cucurbits between northern and southern regions of India.

First reports of Beet curly top virus, Citrus yellow vein-associated virus, and Hop latent viroid in industrial hemp (Cannabis sativa) in Washington State.

In 2021 and 2022, virus-like symptoms were observed in several cultivars of industrial hemp (Cannabis sativa) in two fields in central Washington, USA. Affected plants had a range of symptoms at different developmental stages, with young plants having severe stunting with shortened internodes and reduced flower mass. Young leaves of infected plants also showed light green to total yellowing, and twirling with twisting margins (Fig. S1). Infections of older plants caused less foliar symptoms that consisted of mosaic, mottling, and mild chlorosis on a few branches with tacoing of older leaves. To assess if symptomatic hemp plants were infected with Beet curly top virus (BCTV) as reported earlier (Giladi et al., 2020; Chiginsky et al., 2021), symptomatic leaves were collected from 38 plants, and the extracted total nucleic acids tested by PCR to amplify a 496-base pair (bp) fragment specific to BCTV coat protein (CP) using primers BCTV2-F 5'-GTGGATCAATTTCCAG-ACAATTATC-3' and BCTV2-R 5'-CCCATAAGAGCCATATCA-AACTTC-3' (Strausbaugh et al. 2008). BCTV was found in 37 of the 38 plants. To further assess the virome of symptomatic hemp plants, total RNA was extracted from symptomatic leaves of four plants using Spectrum total RNA isolation kits (Sigma-Aldrich, St. Louis, MO) and subjected to high-throughput sequencing on an Illumina Novaseq platform in paired-end mode (University of Utah, Salt Lake City, UT). The raw reads (33 to 40 million per sample) were trimmed based on quality and ambiguity and resulting paired-end reads of ≈142 bp length were assembled de novo into a pool of contigs (CLC Genomics Workbench 21, Qiagen Inc.). Virus sequences were identified through BLASTn analysis in GenBank (https://www.ncbi.nlm.nih.gov/blast). One contig of 2,929 nucleotides (nt) obtained from one sample (accession no. OQ068391) showed 99.3% identity with BCTV-Wor strain reported from sugar beet in Idaho (accession no. KX867055 Strausbaugh et al., 2017). Another contig of 1,715 nt from a second sample (accession no. OQ068392) shared 97.3% identity with BCTV-CO strain (accession no. KX867022). Two contig sequences of 2,876 nt (accession no. OQ068388) and 1,399 nt (accession no. OQ068389) obtained from the 3rd and 4th samples showed 97.2% and 98.3% identity, respectively, with Citrus yellow vein-associated virus (CYVaV, accession no. MT893740.1) reported in industrial hemp from Colorado (Chiginsky et al., 2021). Contigs of 256 nt sequence (accession no. OQ068390) obtained from the 3rd and 4th samples also showed 99-100% identity with Hop Latent viroid (HLVd) sequences in GenBank (accessions OK143457 and X07397). These results indicated single infections of BCTV strains and co-infection of CYVaV and HLVd in individual plants. To confirm theagents, symptomatic leaves were collected from 28 randomly selected hemp plants and tested by PCR/RT-PCR using primers specific to BCTV (Strausbaugh et al., 2008), CYVaV (Kwon et al., 2021) and HLVd (Matoušek et al., 2001). Amplicons specific to BCTV (496 bp), CYVaV (658 bp) and HLVd (256 bp) were detected in 28, 25, and 2 samples, respectively. BCTV CP sequences obtained by Sanger sequencing from seven samples showed 100% sequence identity with BCTV-CO and BCTV-Wor strains in six and one samples, respectively. Similarly, sequences of CYVaV- and HLVd-specific amplicons showed 100% identity with corresponding sequences in GenBank. To the best of our knowledge, this is the first report of two strains of BCTV (BCTV-CO and BCTV-Wor), CYVaV, and HLVd infecting industrial hemp in Washington state.