Technologies MinION Research Articles

P-2323. Utilizing Nanopore Sequencing Technology to Identify Saporvirus Genotypes in Fecal Samples from Young Children with Diarrhea in Al Ain City, United Arab Emirates

Abstract Background Human sapoviruses are important pathogens causing diarrhea globally. They are increasingly detected in individuals with diarrhea due to the expanded utilization of molecular diagnostic methods. There are 18 genotypes known to infect human (GI.1-7, GII.1-8, GIV.1, and GV.1-2). Notably, the genetic characteristics of sapoviruses circulating in the United Arab Emirates (UAE) remain unclear. This study aims to use next generation sequencing to analyze sapovirus genotypes in stool samples from children under five with diarrhea in Al Ain city. Methods Using multiplex one-step real-time PCR (Allplex™ Gastrointestinal Full Panel Assay), 15 sapovirus-positive stool samples were identified from stool specimens collected for a previous epidemiological study of pathogens causing diarrhea during the period from December 2017 and April 2019. After excluding isolates with low viral load (Ct >30), 8 isolates were used for genotyping studies. Total RNA was extracted using a stool total RNA isolation kit and confirmed by endpoint PCR. Viral RNA was retrotranscribed to cDNA, followed by viral genome amplification, purification, and quantification. After sequencing with Nanopore MinION technology, reads that passed quality control (score >7) were produced. Geneious Prime and Mega11 Software were then used for base-calling, mapping, and phylogenetic analysis. Results PCR amplicon-based platform was developed for sequencing human sapoviruses in order to reduce the cost of sequencing per sample. The system produced successful amplification in 7 out of 8 studied sapovirus-positive samples. All isolates showed close similarity with each other showing more than 99% similarity. Blast analysis of sequences of the amplicons revealed close similarity with East Asian isolates (99%). The closest Middle Eastern isolate was from Iran showing 87% similarity with the UAE isolates. Phylogenetic analysis revealed that seven of the eight sapovirus isolates were found to cluster within the GI.1 genotype. Conclusion Using Nanopore sequencing technology, GI.1 genotype was found to be the most prevalent sapovirus genotype among the studied isolates. The obtained data will enhance our knowledge on the molecular diversity of sapoviruses in the UAE and enrich the current sapoviruses genotyping protocols. Disclosures All Authors: No reported disclosures

Temporal transcriptional profiling of host cells infected by a veterinary alphaherpesvirus using nanopore sequencing

In our research, we performed temporal transcriptomic profiling of host cells infected with Equid alphaherpesvirus 1 (EHV-1) by utilizing direct cDNA sequencing based on nanopore MinION technology. The sequencing reads were harnessed for transcript quantification at various time points. Viral infection-induced differential gene expression was identified through the edgeR package. The identified genes were segmented into six groups based on their kinetic characteristics. The initial three clusters encompass immediate-early response genes, typically transcription factors and elements of antiviral signaling pathways. These genes were either upregulated (cluster 1) or downregulated (clusters 2 and 3) during the early infection phase. The remaining three clusters include late response genes. In these categories, it is challenging to determine whether changes in gene expression are directly connected to the viral infection or merely side effects of the infection. A study of gene associations using the STRINGDB software revealed several gene networks that might be directly impacted by the virus. We also explored whether gene co-expression could be a result of their collective regulation by upstream transcription factors using the Gene Regulatory Network database. Finally, our differential transcript usage (DTU) analysis identified a number of genes that exhibited altered proportions of transcript isoforms in comparison to non-infected cells. Thus, our analysis revealed that EHV-1 infection not only alters host gene expression but also leads to differential use of transcript isoforms, particularly splice variants.

Common Features of Environmental Mycobacterium chelonae from Colorado Using Partial and Whole Genomic Sequence Analyses

Nontuberculous mycobacteria (NTM) are environmentally acquired opportunistic pathogens that cause chronic lung disease in susceptible individuals. While presumed to be ubiquitous in built and natural environments, NTM environmental studies are limited. While environmental sampling campaigns have been performed in geographic areas of high NTM disease burden, NTM species diversity is less defined among areas of lower disease burden like Colorado. In Colorado, metals such as molybdenum have been correlated with increased risk for NTM infection, yet environmental NTM species diversity has not yet been widely studied. Based on prior regression modeling, three areas of predicted high, moderate, and low NTM risk were identified for environmental sampling in Colorado. Ice, plumbing biofilms, and sink tap water samples were collected from publicly accessible freshwater sources. All samples were microbiologically cultured and NTM were identified using partial rpoB gene sequencing. From these samples, areas of moderate risk were more likely to be NTM positive. NTM recovery from ice was more common than recovery from plumbing biofilms or tap water. Overall, nine different NTM species were identified, including clinically important Mycobacterium chelonae. MinION technology was used to whole genome sequence and compare mutational differences between six M. chelonae genomes, representing three environmental isolates from this study and three other M. chelonae isolates from other sources. Drug resistance genes and prophages were common findings among environmentally derived M. chelonae, promoting the need for expanded environmental sampling campaigns to improve our current understanding of NTM species abundance while opening new avenues for improved targeted drug therapies.

Direct metagenomic and amplicon-based Nanopore sequencing of French human monkeypox from clinical specimen.

We report the whole-genome sequence of monkeypox virus obtained using MinION technology (Oxford Nanopore Technologies) from a French clinical specimen during the 2022 epidemic. Amplicon-based sequencing and shotgun metagenomic approaches were directly applied to the sample.

Using a combination of short- and long-read sequencing to investigate the diversity in plasmid- and chromosomally encoded extended-spectrum beta-lactamases (ESBLs) in clinical Shigella and Salmonella isolates in Belgium.

For antimicrobial resistance (AMR) surveillance, it is important not only to detect AMR genes, but also to determine their plasmidic or chromosomal location, as this will impact their spread differently. Whole-genome sequencing (WGS) is increasingly used for AMR surveillance. However, determining the genetic context of AMR genes using only short-read sequencing is complicated. The combination with long-read sequencing offers a potential solution, as it allows hybrid assemblies. Nevertheless, its use in surveillance has so far been limited. This study aimed to demonstrate its added value for AMR surveillance based on a case study of extended-spectrum beta-lactamases (ESBLs). ESBL genes have been reported to occur also on plasmids. To gain insight into the diversity and genetic context of ESBL genes detected in clinical isolates received by the Belgian National Reference Center between 2013 and 2018, 100 ESBL-producing Shigella and 31 ESBL-producing Salmonella were sequenced with MiSeq and a representative selection of 20 Shigella and six Salmonella isolates additionally with MinION technology, allowing hybrid assembly. The bla CTX-M-15 gene was found to be responsible for a rapid rise in the ESBL Shigella phenotype from 2017. This gene was mostly detected on multi-resistance-carrying IncFII plasmids. Based on clustering, these plasmids were determined to be distinct from the circulating plasmids before 2017. They were spread to different Shigella species and within Shigella sonnei between multiple genotypes. Another similar IncFII plasmid was detected after 2017 containing bla CTX-M-27 for which only clonal expansion occurred. Matches of up to 99 % to plasmids of various bacterial hosts from all over the world were found, but global alignments indicated that direct or recent ESBL-plasmid transfers did not occur. It is most likely that travellers introduced these in Belgium and subsequently spread them domestically. However, a clear link to a specific country could not be made. Moreover, integration of bla CTX-M in the chromosome of two Shigella isolates was determined for the first time, and shown to be related to ISEcp1. In contrast, in Salmonella, ESBL genes were only found on plasmids, of which bla CTX-M-55 and IncHI2 were the most prevalent, respectively. No matching ESBL plasmids or cassettes were detected between clinical Shigella and Salmonella isolates. The hybrid assembly data allowed us to check the accuracy of plasmid prediction tools. MOB-suite showed the highest accuracy. However, these tools cannot replace the accuracy of long-read and hybrid assemblies. This study illustrates the added value of hybrid assemblies for AMR surveillance and shows that a strategy where even just representative isolates of a collection used for hybrid assemblies could improve international AMR surveillance as it allows plasmid tracking.

Retrospective Analysis of the SARS-CoV-2 Infection Profile in COVID-19 Positive Patients in Vitoria da Conquista, Northeast Brazil.

Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) is responsible for causing Coronavirus Disease-2019 (COVID-19), a heterogeneous clinical condition that manifests varying symptom severity according to the demographic profile of the studied population. While many studies have focused on the spread of COVID-19 in large urban centers in Brazil, few have evaluated medium or small cities in the Northeast region. The aims of this study were: (i) to identify risk factors for mortality from SARS-CoV-2 infection, (ii) to evaluate the gene expression patterns of key immune response pathways using nasopharyngeal swabs of COVID-19 patients, and (iii) to identify the circulating SARS-CoV-2 variants in the residents of a medium-sized city in Northeast Brazil. A total of 783 patients infected with SARS-CoV-2 between May 2020 and August 2021 were included in this study. Clinical-epidemiological data from patients who died and those who survived were compared. Patients were also retrospectively divided into three groups based on disease severity: asymptomatic, mild, and moderate/severe. Samples were added to a qPCR array for analyses of 84 genes involved with immune response pathways and sequenced using the Oxford Nanopore MinION technology. Having pre-existing comorbidity; being male; having cardiovascular disease, diabetes, and/or chronic obstructive pulmonary disease; and PCR cycle threshold (Ct) values under 22 were identified as risk factors for mortality. Analysis of the expression profiles of inflammatory pathway genes showed that the greater the infection severity, the greater the activation of inflammatory pathways, triggering the cytokine storm and downregulating anti-inflammatory pathways. Viral genome analysis revealed the circulation of multiple lineages, such as B.1, B.1.1.28, Alpha, and Gamma, suggesting that multiple introduction events had occurred over time. This study's findings help identify the specific strains and increase our understanding of the true state of local health. In addition, our data demonstrate that epidemiological and genomic surveillance together can help formulate public health strategies to guide governmental actions.

PIMGAVir and Vir-MinION: Two Viral Metagenomic Pipelines for Complete Baseline Analysis of 2nd and 3rd Generation Data.

The taxonomic classification of viral sequences is frequently used for the rapid identification of pathogens, which is a key point for when a viral outbreak occurs. Both Oxford Nanopore Technologies (ONT) MinION and the Illumina (NGS) technology provide efficient methods to detect viral pathogens. Despite the availability of many strategies and software, matching them can be a very tedious and time-consuming task. As a result, we developed PIMGAVir and Vir-MinION, two metagenomics pipelines that automatically provide the user with a complete baseline analysis. The PIMGAVir and Vir-MinION pipelines work on 2nd and 3rd generation data, respectively, and provide the user with a taxonomic classification of the reads through three strategies: assembly-based, read-based, and clustering-based. The pipelines supply the scientist with comprehensive results in graphical and textual format for future analyses. Finally, the pipelines equip the user with a stand-alone platform with dedicated and various viral databases, which is a requirement for working in field conditions without internet connection.

Comparative performance of CDC-modified SARS-CoV-2 real-time PCR assay with four different commercial assays: laboratory-based study.

The coronavirus infectious disease (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viruses. The pandemic has emerged as a global public health crisis, and the threat of fast-spreading of the latest variants of the coronavirus (such as omicron, delta) is rampant. Therefore, a fast and reliable diagnostic assay is needed to make the clinical decision for further treatment. The study aims to develop a Centers for Disease and Prevention (CDC)–modified qualitative real-time reverse transcriptase PCR (RT-qPCR) assay and parallel assessment of commercially available RT-qPCR assay (Altona, Seegene, BD, and GBC) to detect SARS-CoV-2. Two hundred nine samples were chosen randomly out of around two hundred thousand samples. The panel consisted of SARS-CoV-2-positive (n = 156) and SARS-CoV-2-negative (n = 52) nasopharyngeal swab specimens for a primary clinical evaluation. Furthermore, 29 positive samples were sequenced using Oxford Nanopore Minion technology. Two hundred nine patient sample data of the cycle threshold (Ct) readings for target genes of five assays are 100% sensitive for Ct values. Mean Ct values for N1, N2, RdRp, S, and E of the positive controls in CDC assay, RealStar®, Allplex, GBC, and SD Biosensor were 17.5 ± 0.49, 16.9 ± 0.51, 20 ± 0.49, 21.7 ± 0.38, and 23.1 ± 0.43, respectively. F test value shows ≥ 1, which was statistically significant. All assays showed an efficiency of < 120% and R squares were < 0.99, which is well above the required threshold value. Thus, when taking the CDC-modified assay as a gold standard, the other four assays demonstrated a p value of 0.0000, concordance at 100%, and a Kappa at 1.000. A maximum-likelihood (ML) tree was constructed and compared based on full-length SARS-CoV-2 with Wuhan isolate. These isolates are closely related to the B.1.617 lineage and reference sequences. Therefore, we conclude that all RT-PCR kits assessed in this study shall be used for routine diagnostics of COVID-19 in patients. Supplementary informationThe online version contains supplementary material available at 10.1007/s00580-022-03356-y.

66 Utilizing Third Generation Sequencing to Identify Pathogen Outbreaks Rapidly and Accurately

Abstract The temporal trend of novel pathogen identification has increased over the last decade, with viruses being the main contributor to the increase. Currently, the NAHLN provides veterinary surveillance and testing procedures for high consequence animal diseases, where in many cases, the detection of these diseases is based on real-time PCR assay (ASF, CSF, FMD, and IAV-S) or pathogen specific antibodies using ELISA methods (PRV). However, genetic shift and drift in virus genomes can lead to failure in detection of novel or emerging pathogens. Therefore, with the current trend of increased viral disease emergence, novel methods are needed to accurately and rapidly identify and characterize viral pathogens independent of knowing genomic information. In this study, we developed a novel diagnostic approach to identifying swine pathogens, using surrogate viruses. We implemented the use of Oxford Nanopore MinION Technology to detect long read sequences in real time. Surrogate viruses were used in place of common pathogens such as African Swine Fever, Classical Swine Fever, Pseudorabies, Foot and Mouth Disease, and Influenza A to investigate rapid detection limits. To increase the complexity of a sample, both DNA and RNA viruses were spiked into tissues were analyzed using Oxford Nanopore MinION Technology. This method can detect both DNA and RNA viruses simultaneously without prior knowledge. Additionally, as sequences can be detected real-time, we were able to confidently detect viral pathogens within a sample within 30 min. Additionally, we have established a library preparation and sequencing protocol as well as an informatic pipeline that could allow clinics and diagnostics centers to identify pathogens in less than 10 hours.

Direct RNA Nanopore Sequencing of SARS-CoV-2 Extracted from Critical Material from Swabs

In consideration of the increasing prevalence of COVID-19 cases in several countries and the resulting demand for unbiased sequencing approaches, we performed a direct RNA sequencing (direct RNA seq.) experiment using critical oropharyngeal swab samples collected from Italian patients infected with SARS-CoV-2 from the Palermo region in Sicily. Here, we identified the sequences SARS-CoV-2 directly in RNA extracted from critical samples using the Oxford Nanopore MinION technology without prior cDNA retrotranscription. Using an appropriate bioinformatics pipeline, we could identify mutations in the nucleocapsid (N) gene, which have been reported previously in studies conducted in other countries. In conclusion, to the best of our knowledge, the technique used in this study has not been used for SARS-CoV-2 detection previously owing to the difficulties in the extraction of RNA of sufficient quantity and quality from routine oropharyngeal swabs. Despite these limitations, this approach provides the advantages of true native RNA sequencing and does not include amplification steps that could introduce systematic errors. This study can provide novel information relevant to the current strategies adopted in SARS-CoV-2 next-generation sequencing.

An inducible transposon mutagenesis approach for the intracellular human pathogen Chlamydia trachomatis

Background: Chlamydia trachomatis is a prolific human pathogen that can cause serious long-term conditions if left untreated. Recent developments in Chlamydia genetics have opened the door to conducting targeted and random mutagenesis experiments to identify gene function. In the present study, an inducible transposon mutagenesis approach was developed for C. trachomatis using a self-replicating vector to deliver the transposon-transposase cassette - a significant step towards our ultimate aim of achieving saturation mutagenesis of the Chlamydia genome. Methods: The low transformation efficiency of C. trachomatis necessitated the design of a self-replicating vector carrying the transposon mutagenesis cassette (i.e. the Himar-1 transposon containing the beta lactamase gene as well as a hyperactive transposase gene under inducible control of the tet promoter system with the addition of a riboswitch). Chlamydia transformed with this vector (pSW2-RiboA-C9Q) were induced at 24 hours post-infection. Through dual control of transcription and translation, basal expression of transposase was tightly regulated to stabilise the plasmid prior to transposition. Results: Here we present the preliminary sequencing results of transposon mutant pools of both C. trachomatis biovars, using two plasmid-free representatives: urogenital strain C. trachomatis SWFP- and the lymphogranuloma venereum isolate L2(25667R). DNA sequencing libraries were generated and analysed using Oxford Nanopore Technologies’ MinION technology. This enabled ‘proof of concept’ for the methods as an initial low-throughput screen of mutant libraries; the next step is to employ high throughput sequencing to assess saturation mutagenesis. Conclusions: This significant advance provides an efficient method for assaying C. trachomatis gene function and will enable the identification of the essential gene set of C. trachomatis. In the long-term, the methods described herein will add to the growing knowledge of chlamydial infection biology leading to the discovery of novel drug or vaccine targets.

An inducible transposon mutagenesis approach for the intracellular human pathogen Chlamydia trachomatis.

Background: Chlamydia trachomatis is a prolific human pathogen that can cause serious long-term conditions if left untreated. Recent developments in Chlamydia genetics have opened the door to conducting targeted and random mutagenesis experiments to identify gene function. In the present study, an inducible transposon mutagenesis approach was developed for C. trachomatis using a self-replicating vector to deliver the transposon-transposase cassette - a significant step towards our ultimate aim of achieving saturation mutagenesis of the Chlamydia genome. Methods: The low transformation efficiency of C. trachomatis necessitated the design of a self-replicating vector carrying the transposon mutagenesis cassette (i.e. the Himar-1 transposon containing the beta lactamase gene as well as a hyperactive transposase gene under inducible control of the tet promoter system with the addition of a riboswitch). Chlamydia transformed with this vector (pSW2-RiboA-C9Q) were induced at 24 hours post-infection. Through dual control of transcription and translation, basal expression of transposase was tightly regulated to stabilise the plasmid prior to transposition. Results: Here we present the preliminary sequencing results of transposon mutant pools of both C. trachomatis biovars, using two plasmid-free representatives: urogenital strain C. trachomatis SWFP- and the lymphogranuloma venereum isolate L2(25667R). DNA sequencing libraries were generated and analysed using Oxford Nanopore Technologies' MinION technology. This enabled 'proof of concept' for the methods as an initial low-throughput screen of mutant libraries; the next step is to employ high throughput sequencing to assess saturation mutagenesis. Conclusions: This significant advance provides an efficient method for assaying C. trachomatis gene function and will enable the identification of the essential gene set of C. trachomatis. In the long-term, the methods described herein will add to the growing knowledge of chlamydial infection biology leading to the discovery of novel drug or vaccine targets.

Whole Genome Sequence of Alternaria alternata, the Causal Agent of Black Spot of Kiwifruit.

Alternaria alternata is a pathogen in a wide range of agriculture crops and causes significant economic losses. A strain of A. alternata (Y784-BC03) was isolated and identified from “Hongyang” kiwifruit and demonstrated to cause black spot infections on fruits. The genome sequence of Y784-BC03 was obtained using Nanopore MinION technology. The assembled genome is composed of 33,869,130bp (32.30Mb) comprising 10 chromosomes and 11,954 genes. A total of 2,180 virulence factors were predicted to be present in the obtained genome sequence. The virulence factors comprised genes encoding secondary metabolites, including non-host-specific toxins, cell wall-degrading enzymes, and major transcriptional regulators. The predicted gene clusters encoding genes for the biosynthesis and export of secondary metabolites in the genome of Y784-BC03 were associated with non-host-specific toxins, including cercosporin, dothistromin, and versicolorin B. Major transcriptional regulators of different mycotoxin biosynthesis pathways were identified, including the transcriptional regulators, polyketide synthase, P450 monooxygenase, and major facilitator superfamily transporters.

Isolation and characterization of a novel bacteriophage WO from Allonemobius socius crickets in Missouri.

Wolbachia are endosymbionts of numerous arthropod and some nematode species, are important for their development and if present can cause distinct phenotypes of their hosts. Prophage DNA has been frequently detected in Wolbachia, but particles of Wolbachia bacteriophages (phage WO) have been only occasionally isolated. Here, we report the characterization and isolation of a phage WO of the southern ground cricket, Allonemobius socius, and provided the first whole-genome sequence of phage WO from this arthropod family outside of Asia. We screened A. socius abdomen DNA extracts from a cricket population in eastern Missouri by quantitative PCR for Wolbachia surface protein and phage WO capsid protein and found a prevalence of 55% and 50%, respectively, with many crickets positive for both. Immunohistochemistry using antibodies against Wolbachia surface protein showed many Wolbachia clusters in the reproductive system of female crickets. Whole-genome sequencing using Oxford Nanopore MinION and Illumina technology allowed for the assembly of a high-quality, 55 kb phage genome containing 63 open reading frames (ORF) encoding for phage WO structural proteins and host lysis and transcriptional manipulation. Taxonomically important regions of the assembled phage genome were validated by Sanger sequencing of PCR amplicons. Analysis of the nucleotides sequences of the ORFs encoding the large terminase subunit (ORF2) and minor capsid (ORF7) frequently used for phage WO phylogenetics showed highest homology to phage WOAu of Drosophila simulans (94.46% identity) and WOCin2USA1 of the cherry fruit fly, Rhagoletis cingulata (99.33% identity), respectively. Transmission electron microscopy examination of cricket ovaries showed a high density of phage particles within Wolbachia cells. Isolation of phage WO revealed particles characterized by 40–62 nm diameter heads and up to 190 nm long tails. This study provides the first detailed description and genomic characterization of phage WO from North America that is easily accessible in a widely distributed cricket species.

Variation in type two taste receptor genes is associated with bitter tasting phenylthiocarbamide consumption in mature Targhee and Rambouillet rams.

Bitter taste perception in sheep can lead to avoidance of specific types of forage, such as sagebrush, which is present on many rangeland grazing systems in the Intermountain West. In humans, bitter taste perception is influenced by variation in several TAS2R genes, including more extensively studied TAS2R38 and TAS2R16. We hypothesize that variation in taste receptor genes in sheep is associated with bitter taste. Therefore, the objective of this study was to examine variation in TAS2R genes in relation to consumption of a bitter tasting compound phenylthiocarbamide (PTC) which determines bitter “taster” and “non-taster” status in humans. Rambouillet and Targhee rams (n = 26) were offered various concentrations of PTC solution (0.2–12.29 mM) and water in a side-by-side presentation during two experiments. Blood was collected for DNA isolation and sequencing. Nineteen TAS2R genes were amplified and sequenced with long read Oxford Nanopore MinION technology. A total of 1,049 single nucleotide polymorphisms (SNPs) and 26 haplotypes were identified in these genes. Of these, 24 SNPs and 11 haplotypes were significantly (P < 0.05) associated with PTC consumption in TAS2R3, TAS2R5, TAS2R8, TAS2R9, TAS2R16, TAS2R31-like, TAS2R38, TAS2R39, and TAS2R42-like. Over 50% of the SNPs resulted in a change in amino acid sequence and several resided in potential regulatory regions, which could have downstream functional consequences and influence bitter taste perception in sheep. Further research is needed to validate these associations and elucidate the mechanisms that link variation in TAS2R genes to bitter taste perception in sheep. This may enable producers to select sheep more likely to consume bitter forage such as sagebrush as a flock and rangeland management strategy.

Next-generation sequencing for cytomegalovirus antiviral resistance genotyping in a clinical virology laboratory

The identification of CMV antiviral drug resistance (AVDR) is a critical diagnostic test for immunocompromised patients with CMV infection and a failure of virologic response on optimal antiviral treatment. We developed a next-generation sequencing (NGS) assay for CMV AVDR testing and compared the AVDR mutations identified by NGS to Sanger sequencing. Retrospective review of CMV AVDR testing requests for UL97 and UL54 at our laboratory from 2014 to 2019 was conducted. NGS was performed on the MinION and compared to Sanger sequencing performed at the national reference laboratory. Analysis of the sequences was completed with a novel cloud bioinformatics platform (BugSeq). Twenty patient samples previously characterized were included for study on the MinION. NGS captured all of the CMV AVDR mutations identified by Sanger, and identified additional mutations in UL97 and/or UL54 in 8/13 (62%) of the samples. An analysis of the depth of coverage at which we no longer detected minority single nucleotide variants (SNVs) detected in the original data was conducted, estimating a recall of 95% at 1800 fold coverage. NGS utilizing MinION technology for the detection of CMV AVDR mutations identified additional minority variants in UL97 and UL54 as compared with Sanger sequencing. Through the application of a bioinformatics pipeline available online, our NGS process eliminates barriers associated with the use of the MinION and NGS in clinical laboratories.

Extended-spectrum cephalosporin resistance in Escherichia coli from broiler chickens raised with or without antibiotics in Ontario, Canada

Extended-spectrum cephalosporin (ESC)-resistant Escherichia coli were isolated from fecal samples enriched in ESC-containing broth over two years from broiler chickens raised with and without therapeutic antimicrobials. Pooled samples were obtained from 13 different farms in Canada over two rearing cycles each. Resistant isolates were screened for blaCMY, blaCTX-M, and blaSHV by PCR, and several isolates were sequenced and assembled using Oxford Nanopore MinION technology. Flocks raised with or without antimicrobials contained similar ESC-resistant E. coli. Some plasmids were found in isolates from both farm types and many shared replicon types (IncI1, F, C, and B/O/K/Z) and resistance determinants. Although the use of cephalosporins has stopped in poultry production in Canada, the prevalence of ESC-resistant E. coli found in this study remained high; therefore, further studies are required to determine routes of transmission and persistence.

First report of Sri Lankan cassava mosaic virus and Cassava Mosaic Disease in Laos.

Cassava (Manihot esculenta Crantz) has been traditionally grown as a subsistence crop in Laos, but in recent years cassava cultivation in this country has expanded and is becoming a 'cash crop' for farmers (Malik et al., 2020). This also means that cassava vegetative seed (stakes) is rapidly multiplied and distributed. One of the most important diseases affecting cassava in the world is the Cassava Mosaic Disease (CMD), caused by several species of begomoviruses and disseminated by infected stakes or vectored by the whitefly Bemisia tabaci (Legg et al., 2014). Sri Lankan cassava mosaic virus (SLCMV), a bipartite begomovirus, is the virus species causing CMD in Southeast Asia (SEA) and is widespread in Cambodia, Vietnam, Thailand and south China (Siriwan et al., 2020). During field surveys on July 12 to 14, 2020, the team in south Laos, surveyed 8 fields along the border with Cambodia, in the southern provinces of Attapeu and Champassack and identified CMD symptoms (Supplementary Figure 1A) in only one of the fields, located at Kong District of the Champassack province (GPS coordinates 13.94325, 105.99102). From these 8 fields, samples were collected from every third plant in an X pattern. Photographs from each sampled plant were taken and uploaded into CIAT's PestDisPlace platform (https://pestdisplace.org), for CMD symptom confirmation (Supplementary Figure 1B). Leaf samples were sent to the laboratory for PCR using primers SLCMV-F 5'-ATGTCGAAGCGACCAGCAGATATAAT-3' and SLCMV-R 5'-TTAATTGCTGACCGAATCGTAGAAG-3' targeting the AV1 gene (Dutt et al., 2005), following the protocol described in Siriwan et al. (2020) and primers SLCMV-B-F1 5'-ACCGGATGGCCGCGCCCCCCTCT-3' and SLCMV-B-606R 5'-CACCTACCCTGTTATCGCTAAG-3' targeting part of the BV1 gene. Out of 60 samples collected for the field in Kong district, eleven (18.3%) resulted PCR positive to SLCMV (to DNA-A and DNA-B) but only four plants (6.7%) showed symptoms of CMD (see Supplementary Figure 1B and 1C). None of the samples in the other seven fields had CMD symptoms nor was SLCMV detected in any of these plants. Furthermore, the presence of CMD symptoms in the old leaves of the plants in the affected field suggests that the virus was introduced with contaminated stakes. The complete bipartite genome of one isolate (Champ1), was amplified by Rolling Circle Amplification and sequenced with the nanopore MinION technology as described by Leiva et al. (2020). The sequences were submitted to GenBank under accession nos MT946533 (DNA-A) and MT946534 (DNA-B). A phylogenetic tree for SLCMV and a link to the open SLCMV Nextstrain map (Hadfield et al., 2018) is included in Supplementary Figure 2. The sequences of the DNA-A and DNA-B components of the Champ1 isolate were nearly identical to those of anisolate of SLCMV from Ratanakiri, Cambodia (99.72% for DNA-A and 99.82 for DNA-B; Wang et al., 2016). Phylogenetic analysis (Supplementary Figure 2), grouped isolate Champ1 with those that form the cluster of SEA isolates that contain the shorter version of the rep gene (Siriwan et al., 2020). This short version of rep present a deletion of 7 amino acids at the C-terminus, which is involved in host responses to SLCMV (Wang et al., 2020). The confirmation of CMD and SLCMV in the border between Laos and Cambodia should be followed by disease containment and management strategies, particularly given that the majority cassava varieties grown in Laos are from neighbor countries, most of which have already reported the presence of CMD. Acknowledgements We thank all staff from the CIAT's Cassava Program and the Plant Protection Center of Laos in Vientiane. We acknowledge financial support from the Australian Centre for International Agricultural Research (ACIAR) and the CGIAR Research Program on Roots, Tubers and Bananas (RTB) (https://www.cgiar.org/funders/).

Draft Genome Sequences of 284 Xanthomonas citri pv. citri Strains Causing Asiatic Citrus Canker.

High-quality Illumina assemblies were produced from 284 Xanthomonas citri pv. citri pathotype A strains mostly originating from the Southwest Indian Ocean region, a subset of which was also sequenced using MinION technology. Some strains hosted chromosomally encoded transcription activator-like effector (TALE) genes, an atypical feature for this bacterium.

Recent advances in the laboratory diagnosis of peste des petits ruminants

Peste des petits ruminants (PPR) is a highly contagious and economically important, viral disease of small ruminants caused by the peste des petits ruminants virus (PPRV), which belongs to the genus Morbilivirus in the family Paramyxoviridae. PPR control is achieved mostly through vaccination and/or slaughter of susceptible animals coupled with clinical or laboratory-based diagnosis. Since clinical signs of PPR are not disease-specific and clinical diagnostics is not reliable, it should be confirmed by laboratory testing. Laboratory confirmation of clinical suspicions is made by detection of PPRV in blood, swabs or post-mortem tissues through classical virus isolation (VI), agar gel immunodiffusion (AGID)/agar gel precipitation test (AGPT), counter-immunoelectrophoresis (CIE), immunoperoxidase test (IPT) or enzyme-linked immunosorbent (ELISA) assays. However, these conventional methods have been superseded by more rapid, sensitive and accurate molecular diagnostic techniques based on the amplification of parts of either nucleocapsid (N) or fusion (F) protein gene, such as RT-PCR, real-time RT-PCR, reverse transcription loop-mediated isothermal amplification (RT-LAMP), reverse transcription recombinase polymerase amplification (RT-RPA) and Oxford nanopore MinION technology. Although these molecular diagnostic assays are accurate, rapid and sensitive, they have to be performed in laboratory settings, and samples must be transported under appropriate conditions from the field to the laboratory, which can delay the confirmation of PPRV infection. The recently developed immunochromatographic lateral flow device (IC-LFD) assay can be used in the field (“pen-side”) without the need for expensive equipment, so a well-established laboratory is not required. The control and eventual eradication of PPR is now one of the top priorities for the Food and Agriculture Organization (FAO) and the World Organization for Animal Health (OIE). In 2015, the international community agreed on a global strategy for PPR eradication, setting 2030 as a target date for elimination of the disease