Metagenomics Analysis of the Wheat Virome Identifies Novel Plant and Fungal-Associated Viral Sequences

Wheat viruses including wheat streak mosaic virus, Triticum mosaic virus, and barley yellow dwarf virus cost substantial losses in crop yields every year. Although there have been extensive studies conducted on these known wheat viruses, currently, there is limited knowledge about all components of the wheat (Triticum aestivum L.) virome. Here, we determined the composition of the wheat virome through total RNA deep sequencing of field-collected leaf samples. Sequences were de novo assembled after removing the host reads, and BLASTx searches were conducted. In addition to the documented wheat viruses, novel plant and fungal-associated viral sequences were identified. We obtained the full genome sequence of the first umbra-like associated RNA virus tentatively named wheat umbra-like virus in cereals. Moreover, a novel bi-segmented putative virus tentatively named wheat-associated vipovirus sharing low but significant similarity with both plant and fungal-associated viruses was identified. Additionally, a new putative fungal-associated tobamo-like virus and novel putative Mitovirus were discovered in wheat samples. The discovery and characterization of novel viral sequences associated with wheat is important to determine if these putative viruses may pose a threat to the wheat industry or have the potential to be used as new biological control agents for wheat pathogens either as wild-type or recombinant viruses.

Full Genome Sequence of a Methanomassiliicoccales Representative Enriched from Peat Soil

The full genome of a Methanomassiliicoccales strain, U3.2.1, was obtained from enrichment cultures of percolation fen peat soil under methanogenic conditions, with methanol and hydrogen as the electron acceptor and donor, respectively. Metagenomic assembly of combined long-read and short-read sequences resulted in a 1.51-Mbp circular genome.

Complete Genome Analysis of a Novel Picorna-Like Virus From a Ladybird Beetle, Cheilomenes Sexmaculata

The ladybird beetle Cheilomenes sexmaculata (family Coccinellidae, order Coleoptera), is a common insect predator of agricultural pests. In this study, the full genome sequence of a novel picorna-like virus, temporarily named “Cheilomenes sexmaculata picorna-like virus 1” (CSPLV1), was identified from C. sexmaculata. The full-length sequence of CSPLV1 was 11,384 nucleotide (nt) in length (excluding the polyA tail) with one predicted open reading frame (ORF) encoding 3727 amino acids, a 13 nt 5' untranslated region (UTR) and a 187 nt 3' UTR. The ORF of CSPLV1 consisted of four distinct domains including an RNA virus helicase domain (3029-3319 nt), a peptidase domain (5555-6121 nt), an RNA-dependent RNA polymerase domain (7154-8101 nt) and a picorna-like coat protein domain (8606-9283nt). Phylogenetic analysis based on the conserved RdRP sequence showed that CSPLV1, together with Wuhan house centipede virus 3, Hypera postica associated virus 1 and Diabrotica undecimpunctata virus 1, formed as an unclassified group which is closely related to the clade Solinviviridae. To the best of our knowledge, CSPLV1 is the first picorna-like virus revealed in C. sexmaculata.

Molecular Identification of Prune Dwarf Virus (PDV) Infecting Sweet Cherry in Canada and Development of a PDV Full-Length Infectious cDNA Clone

Prune dwarf virus (PDV) is a member of ilarviruses that infects stone fruit species such as cherry, plum and peach, and ornamentally grown trees worldwide. The virus lacks an RNA silencing suppressor. Infection by PDV either alone, or its mixed infection with other viruses causes deteriorated fruit marketability and reduced fruit yields. Here, we report the molecular identification of PDV from sweet cherry in the prominent fruit growing region of Ontario, Canada known as the Niagara fruit belt using next generation sequencing of small interfering RNAs (siRNAs). We assessed its incidence in an experimental farm and determined the full genome sequence of this PDV isolate. We further constructed an infectious cDNA clone. Inoculation of the natural host cherry with this clone induced a dwarfing phenotype. We also examined its infectivity on several common experimental hosts. We found that it was infectious on cucurbits (cucumber and squash) with clear symptoms and Nicotiana benthamiana without causing noticeable symptoms, and it was unable to infect Arabidopsis thaliana. As generating infectious clones for woody plants is very challenging with limited success, the PDV infectious clone developed from this study will be a useful tool to facilitate molecular studies on PDV and related Prunus-infecting viruses.

Identification, full-length genome sequencing, and field survey of citrus vein enation virus in Italy

Citrus vein enation virus (CVEV) was described in Spain and then it has been reported in several citrus growing areas of Asia, America and Australia. Here, the occurrence of CVEV in Italy has been documented for the first time. The full genome sequence of a CVEV Italian isolate (14Q) was determined by high-throughput sequencing and the presence of the virus was confirmed by RT-PCR and graft-transmission to indicator plants, from which the virus was recovered six-months post-inoculation. Phylogenetic analysis based on the full-length genome of CVEV isolates from different countries showed that they are phylogenetically related to each other based on their geographic origin, rather than on their host and that the Italian isolate is more closely related to the Spanish isolate than to the other ones. A field survey revealed the presence of CVEV in some areas of Campania region (southern Italy), prevalently infecting lemon trees. In the frame of this survey, kumquat was identified for the first time as a host of CVEV. No symptoms were observed in the field so far. The infection of asymptomatic hosts and the transmission by aphid species present in Italy increase the risk that the virus could further spread.

Full Genome Evolutionary Studies of Wheat Streak Mosaic-Associated Viruses Using High-Throughput Sequencing

Wheat streak mosaic (WSM), a viral disease affecting cereals and grasses, causes substantial losses in crop yields. Wheat streak mosaic virus (WSMV) is the main causal agent of the complex, but mixed infections with Triticum mosaic virus (TriMV) and High plains wheat mosaic emaravirus (HPWMoV) were reported as well. Although resistant varieties are effective for the disease control, a WSMV resistance-breaking isolate and several potential resistance-breaking isolates have been reported, suggesting that viral populations are genetically diverse. Previous phylogenetic studies of WSMV were conducted by focusing only on the virus coat protein (CP) sequence, while there is no such study for either TriMV or HPWMoV. Here, we studied the genetic variation and evolutionary mechanisms of natural populations of WSM-associated viruses mainly in Kansas fields and fields in some other parts of the Great Plains using high-throughput RNA sequencing. In total, 28 historic and field samples were used for total RNA sequencing to obtain full genome sequences of WSM-associated viruses. Field survey results showed WSMV as the predominant virus followed by mixed infections of WSMV + TriMV. Phylogenetic analyses of the full genome sequences demonstrated that WSMV Kansas isolates are widely distributed in sub-clades. In contrast, phylogenetic analyses for TriMV isolates showed no significant diversity. Recombination was identified as the major evolutionary force of WSMV and TriMV variation in KS fields, and positive selection was detected in some encoding genomic regions in the genome of both viruses. Furthermore, the full genome sequence of a second Kansas HPWMoV isolate was reported. Here, we also identified previously unknown WSMV isolates in the Great Plains sharing clades and high nucleotide sequence similarities with Central Europe isolates. The findings of this study will provide more insights into the genetic structure of WSM-associated viruses and, in turn, help in improving strategies for disease management.

Identification and Characterization of the Succinate Dehydrogenase Complex Iron Sulfur Subunit B Gene in the Oriental River Prawn, Macrobrachium nipponense

Previous studies have revealed that SDHB has potential functions in the male sexual differentiation and development in M. nipponense through providing ATP. In this study, the functions of Mn-SDHB were further analyzed in depth using quantitative polymerase chain reaction (qPCR), in situ hybridization, western-blot, and RNA interference (RNAi), combined with the histological observations. The full-genome sequence of Mn-SDHB was 54,608 bp at Chromosome 34, including 7 introns and 6 exons. The full-length cDNA sequence of Mn-SDHB was 1,268 base pairs (bp) long with an open reading frame of 807 bp, encoding for 268 amino acids. The highest expression level of Mn-SDHB in different tissues was observed in the testis, and male prawns at post-larval developmental stage 25 during different developmental stages, indicating that SDHB was potentially involved in the male sexual development in M. nipponense. In situ hybridization and western-blot analysis indicated that SDHB plays essential roles in the testis development. The in situ hybridization analysis also implies the potential roles of Mn-SDHB in ovarian development. The expressions of Mn-IAG were decreased after Mn-SDHB dsRNA injection, indicating SDHB has the positive regulatory effects on IAG in M. nipponese. Thus, SDHB was involved in the mechanism of the male sexual development. The testis development was inhibited, and sperms were rarely observed after 10 days of Mn-SDHB dsRNA injection, indicating SDHB has positive effects on the male sexual development in M. nipponense. This study highlights the functions of SDHB in M. nipponense, which provide new insights for the future studies of the male sexual development in other crustacean species.

Isolation, Identification and Genetic Characteristics of an Uncommon Norovirus Genotype, GIX.1[GII.P15], From Kunming, China

Human norovirus, an RNA virus of the family Caliciviridae, is a common viral pathogen causing acute gastroenteritis of all age groups worldwide. To date, tens of thousands genome sequences of norovirus have been uploaded to NCBI database, more than half of them were epidemic strains of GII.4 or GII.17 genotype. However, sequence information on the non-epidemic norovirus strains remains poorly studied. In this study, an uncommon norovirus genotype, GIX.1[GII.P15], was isolated using Raji cells and the full-genome sequence of the strain was extensively characterized. The norovirus virus particles with a diameter of approximately 30 nm and a morphology of spherical and lace-like appearance were observed by electron microscopy. Viral genome replication in Raji cells were confirmed by real-time quantitative reverse transcription-PCR from viral replication kinetics and passaging experiments of the primary virus. Phylogenetic analysis showed that the strain (KMN1) belonged to the GIX.1[GII.P15] genotype and indicated that no recombination has occurred in this strain thus far. Further compared analysis of the full genome sequence with the consensus sequence of GIX.1[GII.P15] genomes revealed a total of 81 nucleotide substitutions (53 in ORF1, 20 in ORF2, and 8 in ORF3) across the genome, but only 6 substitutions resulted in amino acid changes (3 in ORF1, 1 in ORF2, and 2 in ORF3). Moreover, one amino acid substitution at the 302 amino acid site (P302S) was observed in the P2 domain of the capsid protein, and the site was around one of the predicted conformational epitopes on the VP1 protein structure. The genomic information obtained from the novel strain may extend the understanding of the non-epidemic GIX.1[GII.P15] strains.

Population Snapshot of the Extended-spectrum β-lactamase-producing Escherichia Coli Invasive Strains Isolated From a Hungarian Hospital

Background:This study was carried out to determine the prevalence and the genetic background of extended-spectrum β-lactamase-producing Escherichia coli invasive isolates obtained from a tertiary-care hospital in Budapest, Hungary. MethodsBetween October-November 2018, all invasive ESBL-producing E. coli isolates were collected from Central Hospital of Southern Pest. The antimicrobial susceptibility testing was performed according to the EUCAST guidelines. The possible clonal relationships were investigated by core genome (cg)MLST (SeqSphere+) using whole-genome sequencing (WGS) data of isolates obtained from Illumina 251-bp paired-end sequencing. From WGS data acquired antimicrobial resistance genes and replicon types were retrieved using ResFinder3.1, PlasmidFinder2.1, and pMLST-2.0 online tools.ResultsOverall, 25 E. coli isolates were detected and six proved to be resistant to third-generation cephalosporins. Full genome sequence analysis showed that five E. coli isolates belonged to the ST131 clone: two to C1-M27 subclade with blaCTX-M-27 and three to C2/H30Rx subclade with blaCTX-M-15. One isolate belonged to ST1193 with blaCTX-M-27. According to cgMLST, all C2/H30Rx isolates formed a cluster (≤6 allele differences), while the blaCTX-M-27-producing C1-M27 isolates differed at least 35 alleles from each other. Both C2/H30Rx and C1-M27 ST131 isolates harbored similar antimicrobial resistance gene sets. However, only C2/H30Rx isolates had the qnrB and aac(3)-IIa. All isolates showed resistance against ceftriaxone, cefotaxime, and ciprofloxacin, and the C2/H30Rx isolates were also resistant to gentamicin, tobramycin, and ceftazidime.ConclusionsOut of six third-generation cephalosporins-resistant, invasive E. coli, five belonged to the S131clone. This study indicates, that the C2/H30Rx and C1-M27 subclades of the ST131 appear to be the dominant clones collected in a Hungarian hospital.

Identification and genetic characterization of equine infectious anemia virus in Western Balkans

Background Equine infectious anemia (EIA) is a viral disease, caused by the Equine Infectious Anemia virus (EIAV) belonging to the Retroviridae family, genus Lentivirus. Horses (or equids) infected with EIAV are lifelong carriers and they remain contagious for other horses even in the absence of clinical signs. So far, EIAV infection has been reported among horses in North and South America, France, Germany, Italy, Hungary and Romania, with no publication regarding the presence of EIAV in horses in Serbia. To determine the circulation of EIAV among, approximately, the 5000 horses of the Vojvodina region, northern part of Serbia, 316 serum undergone serological testing for EIA. Then, identification and full genome sequencing using next generation sequencing was performed from one EIA positive horse. Results the 316 sera were tested with 3 different commercial agar gel immunodiffusion (AGID) tests and two different commercial enzyme-linked immunosorbent assay (ELISA). With the three AGID kits, 311 (98.4%) among the 316 tested sera were negative and only five (1.6%) sera were positive for EIA. Some discrepancies were seen for the two ELISA kits tested since one exhibited the same results as AGID test and the second gave 295 sera with negative results, five with a positive result and 16 with doubtful outcome. Phylogenetic analysis performed using the full genome sequence showed that EIAV characterized from a horse in Serbia is different from those identify so fare around the world and form a distinct and separate group together with another EIAV strain. Conclusions This study demonstrate for the first time that EIAV is circulating at a low level in the horse population from the Northern part of Serbia. Interestingly, phylogenetic data indicates that this EIAV from the western Balkan region of Europe belongs to a new cluster.