Partial Genome Research Articles

Mammalian orthoreoviruses exhibit rare genotype variability in genome constellations.

Mammalian orthoreoviruses (reoviruses) are currently classified based on properties of the attachment protein, σ1. Four reovirus serotypes have been identified, three of which are represented by well-studied prototype human reovirus strains. Reoviruses contain ten segments of double-stranded RNA that encode 12 proteins and can reassort during coinfection. To understand the breadth of reovirus genetic diversity and its potential influence on reassortment, the sequence of the entire genome should be considered. While much is known about the prototype strains, a thorough analysis of all ten reovirus genome segment sequences has not previously been conducted. We analyzed phylogenetic relationships and nucleotide sequence conservation for each of the ten segments of more than 60 complete or nearly complete reovirus genome sequences, including those of the prototype strains. Using these relationships, we defined genotypes for each segment, with minimum nucleotide identities of 77-88% for most genotypes that contain several representative sequences. We applied segment genotypes to determine reovirus genome constellations, and we propose implementation of an updated reovirus genome classification system that incorporates genotype information for each segment. For most sequenced reoviruses, segments other than S1, which encodes σ1, cluster into a small number of genotypes and a limited array of genome constellations that do not differ greatly over time or based on animal host. However, a small number of reoviruses, including prototype strain Jones, have constellations in which segment genotypes differ from those of most other sequenced reoviruses. For these reoviruses, there is little evidence of reassortment with the major genotype. Future basic research studies that focus on the most genetically divergent reoviruses may provide new insights into reovirus biology. Analysis of available partial sequences and additional complete reovirus genome sequencing may also reveal reassortment biases, host preferences, or infection outcomes that are based on reovirus genotype.

Chromosome-level reference genome of stinkwort, Dittrichia graveolens (L.) Greuter: A resource for studies on invasion, range expansion, and evolutionary adaptation under global change.

Dittrichia graveolens (L.) Greuter, or stinkwort, is a weedy annual plant within the family Asteraceae. The species is recognized for the rapid expansion of both its native and introduced ranges: in Europe, it has expanded its native distribution northward from the Mediterranean basin by nearly 7 °C latitude since the mid-20th century, while in California and Australia the plant is an invasive weed of concern. Here, we present the first de novo D. graveolens genome assembly (1N = 9 chromosomes), including complete chloroplast (151,013 bp) and partial mitochondrial genomes (22,084 bp), created using Pacific Biosciences HiFi reads and Dovetail Omni-C data. The final primary assembly is 835 Mbp in length, of which 98.1% are represented by 9 scaffolds ranging from 66 to 119 Mbp. The contig N50 is 74.9 Mbp and the scaffold N50 is 96.9 Mbp, which, together with a 98.8% completeness based on the BUSCO embryophyta10 database containing 1,614 orthologs, underscores the high quality of this assembly. This pseudo-molecule-scale genome assembly is a valuable resource for our fundamental understanding of the genomic consequences of range expansion under global change, as well as comparative genomic studies in the Asteraceae.

VirMAP for cancer: Characterization of the intratumoral virome in virally associated cancers and a resource for investigators.

2568 Background: A major barrier to understanding the role of viruses in cancer progression has been the lack of sensitivity of current tools to assemble viral genomes in an untargeted way. Here, we utilize our novel VirMAP algorithm to identify eukaryotic and prokaryotic viruses associated with cancers, using RNA sequencing data from The Cancer Genome Atlas (TCGA). Methods: VirMAP was run on 16 TCGA RNASeq datasets of primary tumor tissues, with a focus on cancers with known viral associations (including cervical, head and neck, liver, stomach, esophagus and ovarian). Previously published survival data were used for exploratory analysis. Overall survival (OS) was assessed using the Kaplan-Meier method, and p-values were calculated using log-rank test. Multivariate analysis (MVA) was done using Cox proportional hazards model to control for age, sex, stage, and histology. Results: Viral sequence recovery (among 537 billion sequencing reads from 6185 patients) was at least equal to previously published algorithms such as Kraken, but with higher taxonomic resolution; overall recovery was 0.0041% from 16 tumor types. Cancers with high viral loads such as cervical cancer demonstrated high recovery rates (0.012% of all reads mapped to viral taxa, followed by 0.004% for liver, 0.0018% head and neck, 0.0016% stomach, 0.0009% esophageal, and 0.0002% ovarian). There was a wide range of viral signature richness, with cervical cancer showing the highest viral read count predominated by 18 different HPV serotypes; in contrast, stomach cancer had lower abundance but contained over 300 unique viral species. Exploratory analysis revealed novel correlations between viral signatures and OS. In cervical cancer, presence of HPV45 predicted worse survival (median OS 837 days vs 4086, p=0.0043); MVA demonstrated hazard ratio (HR) of 5.7 [2.6-11.4, p<0.0001]. In stomach cancer, patients often had more than one viral taxon present (mean 3.7, range 0-18); presence of seven or more unique taxa predicted worse OS (median OS 403 days vs 1153, p=0.0001). MVA showed HR of 1.1 for every additional taxon (p=0.0004). Across all 16 tumor types, the detection of phage virus was associated with worse OS (1073 days vs. 2094 days for non-phage virus and 2131 days for no virus, p<0.0001). Conclusions: VirMAP performs deep characterization of the tumor-associated virome in both high and low abundance settings, with generation of partial genome reconstructions and strain level taxonomic classification including phages. Further work is ongoing to optimize viral read normalization based on genome length, as well as subset analysis with disease-specific modifiers such as histology, race, and exposures. Utilizing publicly available platforms such as TCGA, clinical and correlative published data can be leveraged to better understand the nuanced pleotropic effects of the tumor virome on cancer progression.

Genomic analyses indicate two blue swimmer crab species in Australia, evidence for natural interspecific hybridization and genetic structure within species with implications for fisheries management and stock enhancement

Blue swimmer crab species are widely distributed in Indian and west Pacific Oceans including the coastline around Australia and have been identified as potential candidates for stock enhancement. A recent taxonomic review revealed the presence of two blue swimmer crab species in Australia, namely Portunus armatus which is found around most of the Australian coastline, and Portunus pelagicus which is found in the northern coast where it is sympatric with P. armatus. It is important to understand population structure and genetic diversity of Portunus spp. to efficiently manage stocks. In the present study we investigated species classification and population structure of Portunus spp. in Australia using full and partial genomic information, namely mitochondrial COX1 gene sequences derived from whole genome sequencing and single nucleotide polymorphic (SNP) markers developed from partial genome sequencing. Blue swimmer crab samples were collected from the western (n = 52), northern (n = 17) and eastern (n = 110) coasts of Australia. Genetic diversity and population genetic structure were assessed for within and between region variation. Our genomic results indicated the presence of two blue swimmer crab species in Australia. One, namely, P. pelagicus was found only in Northern Territory (Darwin), and other, namely P. armatus was found around Australia. There was evidence of natural hybridisation between two species in Northern Territory (Darwin) where they were sympatric. P. armatus populations from the western, northern and eastern regions were genetically different although there were no significant genetic differences observed between the Queensland and New South Wales populations nor between the two neighbouring populations in Western Australia. Results found in the present study supports the view that the P. armatus populations in western, northern and eastern regions of Australia should be treated as at least three different stocks. A lack of genetic structuring along the eastern coast suggests blue swimmer crabs can be considered as a single stock along the sampled range at least in relation to fisheries enhancement, release programs and broodstock management.

Survey on Betanodavirus in Wild Fish Species in the Turkish Coastlines of the Mediterranean Sea and Molecular Characterization: detecting different genotypes from different fish in different locations

Viral nervous necrosis is a viral disease caused by betanodavirus causing deaths and epidemics in wild or farmed sea fish, especially in juveniles. Betanodavirus has been detected in farmed European seabass and gilthead seabream in Turkey. However, there are no studies on the presence of betanodavirus in wildlife along the Turkish Mediterranean coast. This study aimed to investigate the presence of betanodavirus in wild fish along the Turkish coast of the Mediterranean and to characterize isolates using molecular tools. Hence, 400 fish belonging to 27 different wild species were caught and tested using real-time RT‒PCR. The betanodavirus genome was detected in red mullets (25/50) and garfish (2/4) and identified as RGNNV and RGNNV/SJNNV genotype, respectively according to partial genome sequencing of the RNA1 and RNA2 segments. Phylogenetic analysis showed that isolated viruses were similar to isolates collected from other parts of the Mediterranean Sea and China, India, and Japan. The results of the studies show that different genotypes are circulating in the studied region in different species. These findings indicates that more research on wildlife, both around the farm and in other regions, are needed to prevent spreading of the virus to aquaculture facilities.

Genetic characterization of dengue virus serotype 1 circulating in Reunion Island, 2019–2021, and the Seychelles, 2015–2016

BackgroundAn unprecedent increase in the number of cases and deaths reported from dengue virus (DENV) infection has occurred in the southwestern Indian ocean in recent years. From 2017 to mid-2021 more than 70,000 confirmed dengue cases were reported in Reunion Island, and 1967 cases were recorded in the Seychelles from 2015 to 2016. Both these outbreaks displayed similar trends, with the initial circulation of DENV-2 which was replaced by DENV-1. Here, we aim to determine the origin of the DENV-1 epidemic strains and to explore their genetic characteristics along the uninterrupted circulation, particularly in Reunion.MethodsNucleic acids were extracted from blood samples collected from dengue positive patients; DENV-1 was identified by RT-qPCR. Positive samples were used to infect VERO cells. Genome sequences were obtained from either blood samples or infected-cell supernatants through a combination of both Illumina or MinION technologies.ResultsPhylogenetic analyses of partial or whole genome sequences revealed that all DENV-1 sequences from Reunion formed a monophyletic cluster that belonged to genotype I and were closely related to one isolate from Sri Lanka (OL752439.1, 2020). Sequences from the Seychelles belonged to the same major phylogenetic branch of genotype V, but fell into two paraphyletic clusters, with greatest similarity for one cluster to 2016–2017 isolate from Bangladesh, Singapore and China, and for the other cluster to ancestral isolates from Singapore, dating back to 2012. Compared to publicly available DENV-1 genotype I sequences, fifteen non-synonymous mutations were identified in the Reunion strains, including one in the capsid and the others in nonstructural proteins (NS) (three in NS1, two in NS2B, one in NS3, one in NS4B, and seven in NS5).ConclusionIn contrast to what was seen in previous outbreaks, recent DENV-1 outbreaks in Reunion and the Seychelles were caused by distinct genotypes, all likely originating from Asia where dengue is (hyper)endemic in many countries. Epidemic DENV-1 strains from Reunion harbored specific non-synonymous mutations whose biological significance needs to be further investigated.

Genomic Epidemiology of a Severe Acute Respiratory Syndrome Coronavirus 2 Outbreak in a US Major League Soccer Club: Was It Travel Related?

Professional soccer athletes are at risk of acquiring severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). United States Major League Soccer (MLS) uses protocol-based SARS-CoV-2 testing for identification of individuals with coronavirus disease 2019. Per MLS protocol, fully vaccinated players underwent SARS-CoV-2 real-time polymerase chain reaction testing weekly; unvaccinated players were tested every other day. Demographic and epidemiologic data were collected from individuals who tested positive, and contact tracing was performed. Whole genome sequencing (WGS) was performed on positive specimens, and phylogenetic analyses were used to identify potential transmission patterns. In the fall of 2021, all 30 players from 1 MLS team underwent SARS-CoV-2 testing per protocol; 27 (90%) were vaccinated. One player who had recently traveled to Africa tested positive for SARS-CoV-2; within the following 2 weeks, 10 additional players and 1 staff member tested positive. WGS yielded full genome sequences for 10 samples, including 1 from the traveler. The traveler's sample was Delta sublineage AY.36 and was closely related to a sequence from Africa. Nine samples yielded other Delta sublineages including AY.4 (n = 7), AY.39 (n = 1), and B.1.617.2 (n = 1). The 7 AY.4 sequences clustered together; suggesting a common source of infection. Transmission from a family member visiting from England to an MLS player was identified as the potential index case. The other 2 AY.4 sequences differed from this group by 1-3 nucleotides, as did a partial genome sequence from an additional team member. WGS is a useful tool for understanding SARS-CoV-2 transmission dynamics in professional sports teams.

Molecular Characterization of Avian Rotaviruses F and G Detected in Brazilian Poultry Flocks.

Avian rotaviruses (RVs) are important etiologic agents of gastroenteritis in birds. In general, avian RVs are understudied; consequently, there is a paucity of information regarding these viruses. Therefore, the characterization of these viral species is highly relevant because more robust information on genetic, epidemiologic, and evolutionary characteristics can clarify the importance of these infections, and inform efficient prevention and control measures. In this study, we describe partial genome characterizations of two avian RV species, RVF and RVG, detected in asymptomatic poultry flocks in Brazil. Complete or partial sequences of at least one of the genomic segments encoding VP1, VP2, VP4, VP6, VP7, NSP1, NSP4, NSP4, or NSP5 of 23 RVF and 3 RVG strains were obtained, and demonstrated that multiple variants of both RVF and RVG circulate among Brazilian poultry. In this study, new and important information regarding the genomic characteristics of RVF and RVG is described. In addition, the circulation of these viruses in the study region and the genetic variability of the strains detected are demonstrated. Thus, the data generated in this work should help in understanding the genetics and ecology of these viruses. Nonetheless, the availability of a greater number of sequences is necessary to advance the understanding of the evolution and zoonotic potential of these viruses.

Diversity and phylogeography of begomoviruses and DNA satellites associated with the leaf curl and mosaic disease complex of eggplant

Eggplant is one of the important vegetable crops grown across the world, and its production is threatened by both biotic and abiotic stresses. Diseases caused by viruses are becoming major limiting factors for its successful cultivation. A survey for begomovirus-like symptoms in 72 eggplant fields located in six different Indian states revealed a prevalence of disease ranging from 5.2 to 40.2%, and the symptoms recorded were mosaic, mottling, petiole bending, yellowing, and upward curling, vein thickening, and enation of the leaves, and stunting of plants. The causal agent associated with these plants was transmitted from infected leaf samples to healthy eggplant seedlings via grafting and whiteflies (Bemisia tabaci). The presence of begomovirus was confirmed in 72 infected eggplant samples collected from the surveyed fields exhibiting leaf curl and mosaic disease by PCR using begomovirus specifc primers (DNA-A componet), which resulted in an expected amplicon of 1.2 kb. The partial genome sequence obtained from amplified 1.2 kb from all samples indicated that they are closely related begomovirus species, tomato leaf Karnataka virus (ToLCKV, two samples), tomato leaf curl Palampur virus (ToLCPalV, fifty eggplant samples), and chilli leaf curl virus (ChLCuV, twenty samples). Based on the partial genome sequence analysis, fourteen representative samples were selected for full viral genome amplification by the rolling circle DNA amplification (RCA) technique. Analyses of fourteen eggplant isolates genome sequences using the Sequence Demarcation Tool (SDT) indicated that one isolate had the maximum nucleotide (nt) identity with ToLCKV and eight isolates with ToLCPalV. Whereas, four isolates four isolates (BLC1-CH, BLC2-CH, BLC3-CH, BLC4-CH) are showing nucleotide identity of less than 91% with chilli infecting viruses begomoviruses with chilli infecting begomoviruses and as per the guidelines given by the ICTV study group for the classification of begomoviruses these isolates are considered as one novel begomovirus species, for which name, Eggplant leaf curl Chhattisgarh virus (EgLCuChV) is proposed. For DNA-B component, seven eggplant isolates had the highest nt identity with ToLCPalV infecting other crops. Further, DNA satellites sequence analysis indicated that four betasatellites identified shared maximum nucleotide identity with the tomato leaf curl betasatellite and five alphasatellites shared maximum nucleotide identity with the ageratum enation alphasatellite. Recombination and GC plot analyses indicated that the bulk of begomovirus genome and associated satellites presumably originated from of previously known mono and bipartite begomoviruses and DNA satellites. To the best of our knowledge, this is India's first report of ToLCKV and a noval virus, eggplant leaf curl Chhattisgarh virus associated with eggplant leaf curl disease.

Bioinformatic, genetic and molecular analysis of several badnavirus sequences integrated in the genomes of diverse cocoa (Theobroma cacao L.) germplasm

Endogenous viral elements (EVEs) are integrations of whole or partial viral genomes into the host genome, where they act as host alleles. They exist in a wide range of plant species including Theobroma cacao, the source of chocolate. Because of the international transfer of cacao germplasm, it is important to discriminate between the presence of these inserts and any episomal viruses that may be present in the material. This study was designed to survey a wide range of cacao germplasm, to assess the number, length, orientation, and precise location of the inserts and to identify any effect on the transcription of the gene into which they are inserted. Using a combination of bioinformatic, genetic and molecular approaches, we cloned and sequenced a series of different inserts, including one full-length virus sequence. We also identified, for the first time, an inhibitory effect of the insert on the expression of host genes. Such information is of practical importance in determining the regulation of germplasm transfer and of fundamental relevance to aiding an understanding of the role that such inserts may have on the performance of the host plant.

Abstract 641: VirMAP for cancer: Characterization of the intratumoral virome in virally-associated cancers and a resource for investigators

Abstract Background: A major barrier to understanding the role of viruses in cancer progression has been the lack of sensitivity of current tools to identify and assemble viral genomes in an untargeted way. Here, we utilize our novel VirMAP algorithm to identify eukaryotic and prokaryotic viruses associated with cancers, using RNA sequencing data from The Cancer Genome Atlas (TCGA). Methods: VirMAP was used to analyze TCGA RNA-Seq data derived from primary tumor tissues, with a focus on cancers with known viral associations (cervical, ovarian, head and neck, liver, stomach and esophagus). Previously published survival data were matched with viral signatures for exploratory analysis. Ancom-BC, a differential abundance pipeline, was utilized to screen for viruses which drive outcome. Overall survival (OS) was assessed using the Kaplan-Meier method, and p-values were calculated using log-rank test. Multivariate analysis (MVA) was done using the Cox proportional hazards model to control for age, sex, stage, and histology. Results: Viral sequence recovery (among 723 billion sequencing reads from 2090 patients) was at least equal to previously published algorithms such as Kraken, but with higher taxonomic resolution. Cancers with high viral loads such as cervical cancer demonstrated high recovery rates (0.012% of all sequencing reads mapped to viral taxa, followed by 0.004% for liver, 0.0018% for head and neck squamous cell, 0.0016% for stomach, 0.0009% for esophageal, and 0.0002% for ovarian). There was a wide range of viral signature richness, with cervical cancer showing the highest viral read count predominated by HPV serotypes; in contrast, stomach cancer had lower abundance but contained over 300 unique viral taxa. Exploratory clinical analysis revealed novel correlations between viral signatures and OS. In cervical cancer, presence of HPV45 predicted worse survival (median OS 837 days vs 4086, p=0.0043); MVA demonstrated hazard ratio (HR) of 5.7 [2.6-11.4, p<0.0001]. In stomach cancer, patients often had more than one viral taxon present (mean 3.7, range 0-18); presence of 7 or more unique taxa predicted worse OS (median OS 403 days vs 1153, p=0.0001). MVA showed HR 1.1 for every additional taxon (p=0.0004). Conclusions: VirMAP performs deep characterization of the tumor-associated virome in both high and low abundance settings, with generation of partial genome reconstructions and strain level taxonomic classification. Utilizing publicly available platforms such as TCGA, clinical and correlative published data can be leveraged to better understand the nuanced pleotropic effects of the tumor virome on cancer progression. Citation Format: Yongwoo D. Seo, Neal Bhutiani, Matthew C. Wong, Ashish V. Damania, Golnaz Morad, Matthew Lastrapes, Alexander J. Lazar, Jennifer A. Wargo, Nadim J. Ajami. VirMAP for cancer: Characterization of the intratumoral virome in virally-associated cancers and a resource for investigators [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 641.

Development and validation of microsatellite markers for Barnyard Millet obtained by partial genome assembly

Barnyard millet (Echinochloa esculenta L.)  is grown for human consumption as well as fodder. Barnyard millet is the second most important small millet after finger millet. An Ion S5 Next Generation Sequencer (NGS) was used to sequence the 400bp DNA library of barnyard millet to obtain a draft genome and for mining of microsatellite markers. The De novo assembly yielded assembled reads of 59,67,79,933 bp with 11,39,481 contigs. A total of 46,157 SSRs were identified from 11,39,481 contigs examined. The number of sequences containing SSR were 41,591 and the number of sequences containing more than 1 SSRs were 3,867.  Fifteen SSR markers were validated among the 30 accessions of barnyard millet. The average percentage polymorphism of markers was 66.54 % with average polymorphism information content (PIC) of 0.28 and average SSR primer index (SPI) of 0.57.  These microsatellite markers can be used for fine linkage mapping and genes/QTLs tagging for genetic improvement of target traits in barnyard millet and related crops.&nbsp

Genetic Characteristics and Phylogeographic Dynamics of Lagoviruses, 1988-2021.

Rabbit haemorrhagic disease virus (RHDV), European brown hare syndrome virus (EBHSV), rabbit calicivirus (RCV), and hare calicivirus (HaCV) belong to the genus Lagovirus of the Caliciviridae family that causes severe diseases in rabbits and several hare (Lepus) species. Previously, Lagoviruses were classified into two genogroups, e.g., GI (RHDVs and RCVs) and GII (EBHSV and HaCV) based on partial genomes, e.g., VP60 coding sequences. Herein, we provide a robust phylogenetic classification of all the Lagovirus strains based on full-length genomes, grouping all the available 240 strains identified between 1988 and 2021 into four distinct clades, e.g., GI.1 (classical RHDV), GI.2 (RHDV2), HaCV/EBHSV, and RCV, where the GI.1 clade is further classified into four (GI.1a-d) and GI.2 into six sub-clades (GI.2a-f). Moreover, the phylogeographic analysis revealed that the EBHSV and HaCV strains share their ancestor with the GI.1, while the RCV shares with the GI.2. In addition, all 2020-2021 RHDV2 outbreak strains in the USA are connected to the strains from Canada and Germany, while RHDV strains isolated in Australia are connected with the USA-Germany haplotype RHDV strain. Furthermore, we identified six recombination events in the VP60, VP10, and RNA-dependent RNA polymerase (RdRp) coding regions using the full-length genomes. The amino acid variability analysis showed that the variability index exceeded the threshold of 1.00 in the ORF1-encoded polyprotein and ORF2-encoded VP10 protein, respectively, indicating significant amino acid drift with the emergence of new strains. The current study is an update of the phylogenetic and phylogeographic information of Lagoviruses that may be used to map the evolutionary history and provide hints for the genetic basis of their emergence and re-emergence.

A hybrid deep learning approach for COVID-19 detection based on genomic image processing techniques

The coronavirus disease 2019 (COVID-19) pandemic has been spreading quickly, threatening the public health system. Consequently, positive COVID-19 cases must be rapidly detected and treated. Automatic detection systems are essential for controlling the COVID-19 pandemic. Molecular techniques and medical imaging scans are among the most effective approaches for detecting COVID-19. Although these approaches are crucial for controlling the COVID-19 pandemic, they have certain limitations. This study proposes an effective hybrid approach based on genomic image processing (GIP) techniques to rapidly detect COVID-19 while avoiding the limitations of traditional detection techniques, using whole and partial genome sequences of human coronavirus (HCoV) diseases. In this work, the GIP techniques convert the genome sequences of HCoVs into genomic grayscale images using a genomic image mapping technique known as the frequency chaos game representation. Then, the pre-trained convolution neural network, AlexNet, is used to extract deep features from these images using the last convolution (conv5) and second fully-connected (fc7) layers. The most significant features were obtained by removing the redundant ones using the ReliefF and least absolute shrinkage and selection operator (LASSO) algorithms. These features are then passed to two classifiers: decision trees and k-nearest neighbors (KNN). Results showed that extracting deep features from the fc7 layer, selecting the most significant features using the LASSO algorithm, and executing the classification process using the KNN classifier is the best hybrid approach. The proposed hybrid deep learning approach detected COVID-19, among other HCoV diseases, with 99.71% accuracy, 99.78% specificity, and 99.62% sensitivity.

Mitochondrial Genomes Assembled from Non-Invasive eDNA Metagenomic Scat Samples in Critically Endangered Mammals.

The abundance of many large-bodied vertebrates, both in marine and terrestrial environments, has declined substantially due to global and regional climate stressors that define the Anthropocene. The development of genetic tools that can serve to monitor population's health non-intrusively and inform strategies for the recovery of these species is crucial. In this study, we formally evaluate whether whole mitochondrial genomes can be assembled from environmental DNA (eDNA) metagenomics scat samples. Mitogenomes of four different large vertebrates, the panda bear (Ailuropoda melanoleuca), the moon bear (Ursus thibetanus), the Java pangolin (Manis javanica), and the the North Atlantic right whale (Eubalaena glacialis) were assembled and circularized using the pipeline GetOrganelle with a coverage ranging from 12x to 480x in 14 out of 18 different eDNA samples. Partial mitochondrial genomes were retrieved from three other eDNA samples. The complete mitochondrial genomes of the studied species were AT-rich and comprised 13 protein coding genes, 22 transfer RNA genes, two ribosomal RNA genes, and a putative D-loop/control region. Synteny observed in all assembled mitogenomes was identical to that reported for specimens of the same and other closely related species. This study demonstrates that it is possible to assemble accurate whole mitochondrial chromosomes from eDNA samples (scats) using forthright bench and bioinformatics workflows. The retrieval of mitochondrial genomes from eDNA samples represents a tool to support bioprospecting, bio-monitoring, and other non-intrusive conservation strategies in species considered 'vulnerable', 'endangered', and/or 'critically endangered' by the IUCN Red List of Threatened Species.

Gathered from the vine: a survey of seven grapevine viruses within New England vineyards.

Vineyards in the Southeastern New England American Viticultural Area were surveyed for the incidence of seven major viruses: grapevine leafroll-associated viruses (GLRaV-1, GLRaV-2, GLRaV-3, and GLRaV-4), grapevine fanleaf virus (GFLV), tomato ringspot virus (ToRSV), and tobacco ringspot virus (TRSV). Viruses were detected by ELISA and confirmed by RT-PCR and Sanger sequencing. Multiple viruses were present in 19 out of the 25 vineyards surveyed between 2018 to 2020. GLRaV-3 (27.59%) was the most prevalent virus followed by GLRaV-4 (14.90%), GLRaV-1 (13.52%), GLRaV-2 (11.03%), ToRSV (6.34%), GFLV (5.24%), and TRSV (2.62%). Furthermore, phylogenetic analyses of the viral partial genome sequences acquired in this study revealed that the grapevine viruses present in this area are diverse, indicating that they may have been introduced from different sources. Our findings stress the need for improving the sanitary status of planting materials to avoid the introduction and dissemination of viruses to vineyards in this important wine-producing region of New England.

An increase in enterovirus D68 circulation and viral evolution during a period of increased influenza like illness, The Johns Hopkins Health System, USA, 2022.

An increase in influenza like illness in children and adolescents at the Johns Hopkins Health system during summer 2022 was associated with increased positivity for enterovirus/ rhinovirus. We sought to characterize the epidemiology and viral evolution of enterovirus D68 (EV-D68). A cohort of remnant respiratory samples tested at the Johns Hopkins Microbiology Laboratory was screened for EV-D68. EV-D68 positives were characterized by whole genome sequencing and viral loads were assessed by droplet digital PCR (ddPCR). Genomic changes and viral loads were analyzed along with patients' clinical presentations. Of 566 screened samples, 126 were EV-D68 (22.3%). The median age of EV-D68 infected patients was four years, a total of 52 required supplemental oxygen (41.3%), and 35 (27.8%) were admitted. Lung disease was the most frequent comorbidity that was associated with hospitalization. A total of 75 complete and 32 partial genomes were characterized that made a new cluster within the B3 subclade that was closest to US genomes from 2018. Amino acid changes within the BC and DE loops were identified from 31 genomes (29%) which correlated with an increase in average viral load in respiratory specimens and the need for supplemental oxygen. EV-D68 outbreaks continue to cause influenza like illness that could be overwhelming for the health system due to a significant demand for high flow oxygen. Viral evolution and an increase in the susceptible population are likely driving the trends of the increased EV-D68 infections.

Identification and Genomic Characterization of Anelloviruses in Patients with Chronic Lymphocytic Leukemia

Purpose. Metagenomics has revealed that, in addition to the digestive tract, certain viruses are also commonly found in human blood. In order to explore and monitor potential novel viruses, three serum samples of patients with chronic lymphocytic leukemia were collected at the No. 2 People’s Hospital of Changshu City, China. Materials and Methods. We sequenced the virome of serum samples from three patients with chronic lymphocytic leukemia using an unbiased viral metagenomic approach and subsequently performed maximum likelihood phylogenetic analysis using MrBayes v3.2. In addition, pairwise sequence comparison was produced with ORF1 amino acid sequences of anelloviruses within Bayesian consensus tree. Results. Partial genomes of eight different anelloviruses containing the complete ORF1 gene have been identified. BLASTp results showed that the amino acid sequence identity of these viruses with the best match in GenBank was between 56.22% and 95.43%. Phylogenetic analysis based on ORF1 indicated that seven sequences belong to the genus Alphatorquevirus and one sequence belongs to the genus Gammatorquevirus. Conclusions. This virological investigation has increased our understanding of the diversity of anelloviruses in human serum, but further study is needed to verify its potential correlation with disease.

Characterization of metagenome-assembled genomes of two endo-archaea of Candida tropicalis

IntroductionHost-microbe interactions are pivotal in host biology, ecology, and evolution. Recent developments in sequencing technologies have provided newer insights into the same through the hologenome concept.MethodsWe report here the study on metagenome-assembled genomes (MAGs) associated with Candida tropicalis (studied through shotgun metagenome sequencing), adding to the knowledge about endomicrobiomes of yeast. De novo assembly and binning recovered two partial archaeal genomes, taxonomically belonging to the phylum Asgardarchaeota.Results and DiscussionThe phylogenomic analysis based on the core genes revealed that both the binned genomes cladded separately with the less studied and uncultivated ‘Candidatus’ superphylum, designated as Asgard archaea (the nearest known relative of eukaryotes). Between the two binned genomes, the average nucleotide index (ANI) was 71.2%. The average nucleotide identities (ANI) of the two binned genomes with ‘Candidatus Heimdallarchaeota’ were 60.4-61.2%. The metabolic pathways of both the binned genomes predicted genes belonging to sulfur reduction, Kreb’s pathway, glycolysis, and C1 carbon metabolism. Further, both the binned genomes were predicted to support autotrophic as well as the heterotrophic mode of growth, which might probably help the host in its nutritional requirements also. Further, the genomes showed few eukaryotic signature proteins (ESPs) and SNARE proteins indicating that members of Asgardarchaeota are the closest relatives of eukaryotes. The gaps present in the metabolic potential of the MAGs obtained and the absence of a few essential pathways shows that they are probably in a symbiotic relationship with the host. The present study, reports for the first-time endosymbiosis of Asgard archaea with yeast. It also provides insights into the metabolic potential, ecology, evolutionary history, and endosymbiotic nature of the important but 160 poorly studied Asgard archaea.

Pathological and molecular studies on elephant endotheliotropic herpesvirus haemorrhagic disease among captive and free-range Asian elephants in India.

In the present research pathology and molecular diagnosis of elephant endotheliotropic herpes virus-haemorrhagic disease (EEHV-HD) among Asian elephants was studied. Out of 76 cases, 20 were positive for EEHV infection in PANPOL and POL1 based semi-nested PCR. Out of 20 samples, 10 samples were fatal cases of EEHV-HD while 10 were of either subclinical or latent infection. Acute onset haemorrhagic disease with EEHV-HD had anorexia, facial and neck swelling, cyanotic buccal mucosa and tongue, nasal and ocular discharge, and colic. The hallmark of gross finding in all cases were severe haemorrhagic lesions in the internal organs viz. cyanosis of tongue with multifocal petechial haemorrhages, diffuse epicardial and endocardial haemorrhages, swollen liver (rounded edges) with parenchymal haemorrhages, serosal and mucosal haemorrhages in gastrointestinal tract, congested kidneys with corticomedullary haemorrhages, highly congested meninges, and brain capillaries with haemorrhages. Microscopic findings in all the cases had severe vascular changes in the visceral organs. Microthrombi was present in the vasculature of tongue, heart, lung, liver, kidney, and brain. The endothelial lining of most of the blood vessels were swollen with apoptotic changes. Amphophilic to basophilic intranuclear inclusion bodies were observed in the endothelial cells. Immunostaining using anti-EEHV DNAPOL hyperimmune sera revealed intense positive signals in the endothelium of blood vessels and their walls. Quantification of viral load in necropsy tissue samples revealed highest in the heart (7.4×106/μg of sample) and least in the brain (9×103/μg of sample). The PCR amplicons from EEHV1 specific genes (POL1(U38) and TER were subjected to partial genome sequencing which had 99.9% similarity with the EEHV1A subtype. It was concluded that Asian elephants in India are latently infected for EEHV1 and in all the fatal EEHV-HD cases, EEHV1A subtype was the causative agent with characteristic pathomorphological changes in visceral organs.