Complete Coding Sequence Research Articles

Whole Genome Sequencing and Phylogenetic Analysis of Rabies Viruses from Bats in Connecticut, USA, 2018-2019.

We performed whole genome sequencing and genetic characterization of rabies viruses (RABV) detected in bats submitted to the Connecticut Veterinary Medical Diagnostic Laboratory (CVMDL) during 2018–2019. Among 88 bats submitted to CVMDL, six brain samples (6.8%, 95% confidence interval: 1.6% to 12.1%) tested positive by direct fluorescent antibody test. RABVs were detected in big brown bats (Eptesicus fuscus, n = 4), a hoary bat (Lasiurus cinereus, n = 1), and an unidentified bat species (n = 1). Complete coding sequences of four out of six detected RABVs were obtained. In phylogenetic analysis, the RABVs (18-62, 18-4347, and 19-2274) from big brown bats belong to the bats EF-E1 clade, clustering with RABVs detected from the same bat species in Pennsylvania and New Jersey. The bat RABV (19-2898) detected from the migratory hoary bat belongs to the bats LC clade, clustering with the eleven viruses detected from the same species in Arizona, Washington, Idaho, and Tennessee. The approach used in this study generated novel data regarding genetic relationships of RABV variants, including their reservoirs, and their spatial origin and it would be useful as reference data for future investigations on RABV in North America. Continued surveillance and genome sequencing of bat RABV would be needed to monitor virus evolution and transmission, and to assess the emergence of genetic mutations that may be relevant for public health.

Field surveys in Croatia and North Macedonia reveal two novel phleboviruses circulating in sandflies.

Sandfly-borne phleboviruses are distributed widely throughout the Mediterranean Basin, presenting a threat to public health in areas where they circulate. However, the true diversity and distribution of pathogenic and apathogenic sandfly-borne phleboviruses remains a key issue to be studied. In the Balkans, most published data rely on serology-based studies although virus isolation has occasionally been reported. Here, we report the discovery of two novel sandfly-borne phleboviruses, provisionally named Zaba virus (ZABAV) and Bregalaka virus (BREV), which were isolated in Croatia and North Macedonia, respectively. This constitutes the first isolation of phleboviruses in both countries. Genetic analysis based on complete coding sequences indicated that ZABAV and BREV are distinct from each other and belong to the genus Phlebovirus, family Phenuiviridae. Phylogenetic and amino acid modelling of viral polymerase shows that ZABAV and BREV are new members of the Salehabad phlebovirus species and the Adana phlebovirus species, respectively. Moreover, sequence-based vector identification suggests that ZABAV is mainly transmitted by Phlebotomus neglectus and BREV is mainly transmitted by Phlebotomus perfiliewi. BREV neutralizing antibodies were detected in 3.3% of human sera with rates up to 16.7% in certain districts, demonstrating that BREV frequently infects humans in North Macedonia. In vitro viral growth kinetics experiments demonstrated viral replication of both viruses in mammalian and mosquito cells. In vivo experimental studies in mice suggest that ZABAV and BREV exhibit characteristics making them possible human pathogens.

Incidential Findings of Mutations in the PIGA Gene Are Highly Specific for the Presence of PNH Clones

Background: Paroxysmal nocturnal hemoglobinuria (PNH) is a hemolytic anemia associated with severe thrombophilia and characterized by complement-mediated lysis of erythrocytes lacking glycosylphosphatidylinositol (GPI)-anchored proteins. In the majority of cases, GPI deficiency is caused by somatic mutations in the PIGA gene. Presence of PNH clones is associated with acquired aplastic anemia (AA) and can be found in patients with myelodysplastic syndrome (MDS) or rarely other myeloid neoplasms (MN). Flow cytometric analysis for deficiency of GPI-anchored proteins on multiple cell lineages detects PNH clones, and PIGA mutational analysis is not mandatory to establish the diagnosis. In contrast, molecular genetic analysis of targeted gene panels is widely used in the diagnostic workup of MN. We hypothesized that the inclusion of PIGA into the myeloid gene panel could identify obscure cases with PNH clones irrespective of the initial clinical suspicion.Aim: To assess the significance of incidental findings of mutations in PIGA in the diagnostic workup of MN.Methods: 20,320 consecutive patients undergoing sequencing analysis for a confirmed or suspected MN were analyzed for the presence of mutations in the PIGA gene. Patients with confirmed PNH analyzed only for PIGA were not included, and cases with previously known PIGA mutations were excluded from further analysis. DNA was isolated from peripheral blood (PB) or bone marrow, and sequencing was performed on NovaSeq after Illumina DNA Prep for Enrichment library preparation (Illumina, San Diego, CA) and hybrid capture of a 41 gene panel including the complete coding sequence of PIGA (IDT Inc., Coralville, IA); data was analyzed with Pisces and Pindel (BaseSpace, Illumina). Flow cytometry was performed on granulocytes, monocytes, and erythrocytes in PB using antibodies against GPI-anchored proteins (CD14, CD24, CD55, and CD59), fluorescein-labeled proaerolysin (FLAER) staining, and Navios cytometers; analysis was done using Kaluza software (both Beckman Coulter, Miami, FL).Results: PIGA mutations were newly identified in 67 patients (0.3%) undergoing targeted sequencing within the diagnostic workup of MN. 30 patients were excluded from further analysis as the gene panel had been requested for a MN associated with previously diagnosed PNH. From the remaining patients, PB for flow cytometry analysis could be obtained from 20 patients. Flow cytometry confirmed the presence of a PNH clone in 17 (85%) of these patients (median clone size: 41% for granulocytes, 54.5% for monocytes, and 12% for erythrocytes). In 3 patients (15%) with unexpected PIGA mutations, flow cytometry detected no PNH clone. The type of PIGA mutations differed significantly in those cases: Patients in whom a PNH clone was confirmed, showed protein-truncating frame-shift (41%) or nonsense (6%) mutations, splice site mutations (18%), or multiple mutations (35%) including at least one protein-truncating mutation at a median variant allele frequency (VAF) of 15.4% (range 2.0% to 50.1%). In contrast, patients without a PNH clone showed only singular missense mutations of PIGA with a VAF of 3.6% to 5.3% (Figure 1). Final diagnoses in patients with confirmed clones were sole PNH (n=9), or PNH clone associated with MDS (n=4), AA (n=3), and AML (n=1), and additional mutations in other genes were observed in 9 cases. While the initial clinical presentation included the differential diagnosis of PNH in some of the patients, flow cytometry was requested as a direct result of the PIGA mutation in 4 cases with an accompanying MN and in 3 patients without - the later showing a median latency of 6.5 years from the initial clinical presentation to the diagnosis.Conclusions: The inclusion of PIGA into a standardized targeted sequencing panel for MN helps to identify patients with PNH clones irrespective of the initial clinical suspicion but is not sufficient to rule out PNH. Protein-truncating PIGA mutations are highly specific for PNH clones whereas singular missense mutations may not necessarily effect GPI biosynthesis. Our data indicate that the incidental finding of a PIGA mutation in sequencing analysis shall entail flow cytometry of GPI-anchored proteins in PB. The potential clinical sequelae and the availability of specific treatment options such as complement inhibitors warrant the thorough exclusion of PNH in the diagnostic workup of suspected MN. [Display omitted] DisclosuresHoermann: Novartis: Honoraria. Haferlach: MLL Munich Leukemia Laboratory: Other: Part ownership. Haferlach: MLL Munich Leukemia Laboratory: Other: Part ownership. Kern: MLL Munich Leukemia Laboratory: Other: Part ownership.

Molecular cloning, functional characterization, tissue expression and polymorphism analysis of buffalo PRDX6 gene

PRDX6 is a bifunctional protein involved in antioxidant regulation and phospholipid metabolism. Previous studies have shown that PRDX6 is involved in some biological pathways and networks related to lactation. The aim of this study was to explore the characteristics, function, tissue expression and variation of buffalo PRDX6 gene. We cloned and characterized the complete coding sequence (CDS) of buffalo PRDX6. The CDS of PRDX6 for swamp and river buffalo is the same, which consists of 675 nucleotides and encodes a protein of 224 amino acids. Buffalo PRDX6 contains one PRX_1cys functional domain (AA 7–222), which is probably related to the regulation of oxidative stress. Multi-tissue differential expression analysis showed that buffalo PRDX6 was highly expressed in the muscle, brain, lung and small intestine during non-lactation and lactation, and there were significant differences in expression in all the tissues except the small intestine between the two periods. It is worth noting that the mRNA abundance of buffalo PRDX6 in non-lactating mammary gland is higher than that in lactating mammary gland. Among the two single nucleotide polymorphisms (SNPs) identified in the CDS in this study, c.261C>T is shared by the two types of buffalo with different allelic frequencies, and c.426T>G is found only in river buffalo. The c.426T>G is non-synonymous, resulting in the amino acid substitution p.Asn142Lys. Only one nucleotide differential site is identified in PRDX6 gene between buffalo and other species of Bovidae. Phylogenetic analysis indicated that buffalo PRDX6 has a closer genetic relationship with that of the species in Bovidae. These results indicate that PRDX6 probably plays a crucial role in the mammary gland of buffalo. This study provides the foundation for further functional studies of PRDX6 in buffalo.

European subtype of tick-borne encephalitis virus. Literature review

This review is devoted to the European subtype of tick-borne encephalitis virus (TBEV). It summarizes and analyzes the information available at the scientific literature on the genetic and biological properties of strains of this virus subtype. A comparative analysis of the complete coding sequences of all currently recognized tick-borne flaviviruses was carried out. It was noted that the differences in TBEV strains included in the European subtype are minimal, which indicates a higher degree of their genetic homogeneity than in strains of the Far Eastern and Siberian subtypes. The level of differences in the genome of strains of the European subtype, depending on the region and the source of isolation, was analyzed. No relationship was found between the level of homology of nucleotide sequences of TBEV strains of the European subtype and the source of isolation. The proposed models for the evolution of TBE are described. The area of TBE of the European subtype in Eurasia is analyzed. The maps of the geographical distribution of the European subtype are presented. It shows the European subtype TBE is found in 14 regions of Russia. TBE of this subtype, as a rule, causes a disease with a milder course in comparison with TBE caused by a virus of the Far Eastern or Siberian subtypes. An analysis of the main vectors and reservoir hosts of the European subtype TBEV in Europe and in Siberia has been carried out. It is emphasized that in Eurasia the European TBEV circulates in territories that differ significantly in climatic conditions, relief, landscape, and characteristics of biotopes. However, analysis of scientific literature data showed that, despite these differences, it has a high degree of genome stability.

The colorful mantle of the giant clam Tridacna squamosa expresses a homolog of electrogenic sodium: Bicarbonate cotransporter 2 that mediates the supply of inorganic carbon to photosynthesizing symbionts.

Giant clams live in symbiosis with phototrophic dinoflagellates, which reside extracellularly inside zooxanthellal tubules located mainly in the colourful and extensible outer mantle. As symbiotic dinoflagellates have no access to the ambient seawater, they need to obtain inorganic carbon (Ci) from the host for photosynthesis during illumination. The outer mantle has a host-mediated and light-dependent carbon-concentrating mechanism to augment the supply of Ci to the symbionts during illumination. Iridocytes can increase the secretion of H+ through vacuolar H+-ATPase to dehydrate HCO3− present in the hemolymph to CO2. CO2 can permeate the basolateral membrane of the epithelial cells of the zooxanthellal tubules, and rehydrated back to HCO3− in the cytoplasm catalysed by carbonic anhydrase 2. This study aimed to elucidate the molecular mechanism involved in the transport of HCO3− across the apical membrane of these epithelial cells into the luminal fluid surrounding the symbionts. We had obtained the complete cDNA coding sequence of a homolog of electrogenic Na+-HCO3− cotransporter 2 (NBCe2-like gene) from the outer mantle of the fluted giant clam, Tridacna squamosa. NBCe2-like gene comprised 3,399 bp, encoding a protein of 1,132 amino acids of 127.3 kDa. NBCe2-like protein had an apical localization in the epithelial cells of zooxanthellal tubules, denoting that it could transport HCO3− between the epithelial cells and the luminal fluid. Furthermore, illumination augmented the transcript level and protein abundance of NBCe2-like gene/NBCe2-like protein in the outer mantle, indicating that it could mediate the increased transport of HCO3− into the luminal fluid to support photosynthesis in the symbionts.

Molecular characterization of superoxide dismutase and catalase genes, and the induction of antioxidant genes under the zinc oxide nanoparticle-induced oxidative stress in air-breathing magur catfish (Clarias magur).

The deduced amino acid sequences from the complete cDNA coding sequences of three antioxidant enzyme genes (sod1, sod2, and cat) demonstrated that phylogenetically the magur catfish (Clarias magur) is very much close to other bony fishes with complete conservation of active site residues among piscine, amphibian, and mammalian species. The three-dimensional structures of three antioxidant enzyme proteins are very much similar to mammalian counterparts, thereby suggesting the functional similarities of these enzymes. Exposure to ZnO NPs resulted in an oxidative stress as evidenced by an initial sharp rise of intracellular concentrations of hydrogen peroxide (H2O2) and malondialdehyde (MDA) but decreased gradually at later stages. The level of glutathione (GSH) also increased gradually in all the tissues examined after an initial decrease. Biochemical and gene expression analyses indicated that the magur catfish has the ability to defend the ZnO NP-induced oxidative stress by inducing the SOD/CAT enzyme system and also the GSH-related enzymes that are mediated through the activation of various antioxidant-related genes both at the transcriptional and translational levels in various tissues. Furthermore, it appeared that the stimulation of NO, as a consequence of induction nos2 gene, under NP-induced oxidative stress serves as a modulator to induce the SOD/CAT system in various tissues of magur catfish as an antioxidant strategy. Thus, it can be contemplated that the magur catfish possesses a very efficient antioxidant defensive mechanisms to defend against the oxidative stress and also from related cellular damages during exposure to ZnO NPs into their natural environment.

Identification of novel polymorphism and in silico analysis of caprine DNAJB3 gene

• DNAJB3, a molecular chaperon, was analysed first time both in vivo and in silico in goat. • In the amplified complete coding sequence of caprine DNAJB3, one nucleotide substitution at 419 th (C > A) position was identified. • In silico structural analysis predicted change in amino acid (Ala > Glu) at 140 th position with minor difference (7%) in three dimensional protein structure. • Computational analysis showed differential interaction of the wild and mutant type DNAJB3 proteins with other stress related proteins. DNAJB3 acts as a cochaperon and stimulates ATPase activity of HSP family genes through its J domain. An in vivo and in silico study was conducted to get comprehensive idea on the role of DNAJB3 gene in goat. The coding sequence of DNAJB3 gene was amplified and searched for polymorphism in three indigenous goat (i.e. Black Bengal, Ganjam and Raighar) populations. One nucleotide substitution at 419th (C > A) position (wild and mutant) with resultant change in amino acid (Ala > Glu) at 140th position was identified. The comparative nucleotide sequence analysis showed 68 variable sites that included 53 parismony informative sites and 15 singleton sites. In silico structural analysis revealed minor difference (7%) in three dimensional protein structure between mutant and wild type DNAJB3 proteins. The protein-protein interactions of DNAJB3 with HSP72, JNK stress kinase, and IKKβ stress kinase were also predicted. The mutant (Raighar) type DNAJB3 protein predicted to interact only with A chain of the JNK stress kinase. Both the wild and mutant type variants of DNAJB3 proteins predicted to interact with A and B chain of hexamer IKKβ stress kinase. This study will be helpful to use DNAJB3 as a molecular marker for thermal tolerance in future.

Comparative Analysis of Novel Strains of Porcine Astrovirus Type 3 in the USA.

Porcine astrovirus type 3 (PoAstV3) has been previously identified as a cause of polioencephalomyelitis in swine and continues to cause disease in the US swine industry. Herein, we describe the characterization of both untranslated regions, frameshifting signal, putative genome-linked virus protein (VPg) and conserved antigenic epitopes of several novel PoAstV3 genomes. Twenty complete coding sequences (CDS) were obtained from 32 diagnostic cases originating from 11 individual farms/systems sharing a nucleotide (amino acid) percent identity of 89.74–100% (94.79–100%), 91.9–100% (96.3–100%) and 90.71–100% (93.51–100%) for ORF1a, ORF1ab and ORF2, respectively. Our results indicate that the 5′UTR of PoAstV3 is highly conserved highlighting the importance of this region in translation initiation while their 3′UTR is moderately conserved among strains, presenting alternative configurations including multiple putative protein binding sites and pseudoknots. Moreover, two predicted conserved antigenic epitopes were identified matching the 3′ termini of VP27 of PoAstV3 USA strains. These epitopes may aid in the design and development of vaccine components and diagnostic assays useful to control outbreaks of PoAstV3-associated CNS disease. In conclusion, this is the first analysis predicting the structure of important regulatory motifs of neurotropic mamastroviruses, which differ from those previously described in human astroviruses.

Molecular characterization, cellular localization and light-dependent expression of dinoflagellate vacuolar-type H+-ATPase (VHA) subunit B in the colourful outer mantle of the giant clam, Tridacna squamosa, indicate the involvement of VHA in CO2 uptake in the photosynthesizing symbionts

Giant clams are animal-dinoflagellate associations found in Indo-Pacific reef ecosystems. The clam host obtains organic nutrients from phototrophic dinoflagellates of genera Symbiodinium, Cladocopium, and Durusdinium, which reside extracellularly as symbionts (alias zooxanthellae) in the luminal fluid of zooxanthellal tubules located mainly in the colourful outer mantle. The host also needs to supply the symbionts with inorganic carbon for photosynthesis. Symbiont photosynthesis can be impeded by inhibitors of vacuolar H+-ATPase (VHA) because the host possesses a carbon concentration mechanism consisting of VHA to facilitate the supply of CO2(aq) to the symbionts. Here, we report that VHA was also expressed in dinoflagellates residing in the outer mantle of the fluted giant clam, Tridacna squamosa. Three complete cDNA coding sequences of VHA subunit B (VHA-B), one for each genus of dinoflagellate, had been obtained, and each sequence comprised 1482 bp, encoding a protein of 493 amino acids (~55 kDa). As these three sequences were highly similar, we could only design real-time PCR primers to quantify comprehensively zooxanthellae-VHA-B (Zoox-VHA-B) that represented VHA-B of all three genera of dinoflagellates. The outer mantle had the highest transcript level of Zoox-VHA-B among the three organs studied, and illumination led to a significant increase in the protein abundance of Zoox-VHA-B therein. Zoox-VHA-B was immunolocalized to intracellular vesicles, which could apparently align and fuse with the plasma membrane, in the symbiotic dinoflagellates. Overall, these results indicate that photosynthesizing symbionts could increase the capacity of H+ secretion through VHA-containing vesicles to promote the dehydration of luminal HCO3− and the absorption of CO2(aq) during illumination.

Proposed Update to the Taxonomy of Pestiviruses: Eight Additional Species within the Genus Pestivirus, Family Flaviviridae.

Pestiviruses are plus-stranded RNA viruses belonging to the family Flaviviridae. They comprise several important pathogens like classical swine fever virus and bovine viral diarrhea virus that induce economically important animal diseases. In 2017, the last update of pestivirus taxonomy resulted in demarcation of 11 species designated Pestivirus A through Pestivirus K. Since then, multiple new pestiviruses have been reported including pathogens associated with disease in pigs or small ruminants. In addition, pestivirus sequences have been found during metagenomics analysis of different non-ungulate hosts (bats, rodents, whale, and pangolin), but the consequences of this pestivirus diversity for animal health still need to be established. To provide a systematic classification of the newly discovered viruses, we analyzed the genetic relationship based on complete coding sequences (cds) and deduced polyprotein sequences and calculated pairwise distances that allow species demarcation. In addition, phylogenetic analysis was performed based on a highly conserved region within the non-structural protein NS5B. Taking into account the genetic relationships observed together with available information about antigenic properties, host origin, and characteristics of disease, we propose to expand the number of pestivirus species to 19 by adding eight additional species designated Pestivirus L through Pestivirus S.

DNA binding protein CgIkaros-like regulates the proliferation of agranulocytes and granulocytes in oyster (Crassostrea gigas)

DNA-binding protein Ikaros is a major determinant of haematopoietic lineage, especially in the development, differentiation and proliferation of lymphocytes. In the present study, a Ikaros homologue (designed as CgIkaros-like) was identified and characterized as a vital determinant in the proliferation of haemocytes during haematopoiesis of Pacific oyster Crassostrea gigas. The complete coding sequence of CgIkaros-like was of 1329 bp encoding a predicted polypeptide of 442 amino acids with four ZnF regions, locating at the C-terminus and N-terminus respectively. The highest expression level of CgIkaros-like mRNA was found in gills, followed by haemocytes and gonad. The mRNA transcripts of CgIkaros-like could be detected in all the haemocytes with higher abundance in semi-granulocytes and agranulocytes. CgIkaros-like protein was localized in both of cytoplasm and nucleus with higher abundance in nucleus of oyster haemocytes. The mRNA and protein expression levels of agranulocyte marker CgCD9, granulocyte marker CgAATase, cell cycle related gene CgCDK2, Notch receptor CgNotch and Notch target gene CgHes1 all increased significantly (p < 0.05) after CgIkaros-like was interfered by siRNAs, which were about 27.33-, 2.63-, 24.34-, 4.45- and 6.08-fold of that in the siRNA-NC control group, respectively. While the transcripts of CgGATA3 and CgRunx did not change significantly after CgIkaros-like was interfered. These results demonstrated that CgIkaros-like functioned as a transcription factor combined with Notch pathway to mediate CgCDK2 and regulate the proliferation of oyster haemocytes.

Single-Molecule Real-Time and Illumina-Based RNA Sequencing Data Identified Vernalization-Responsive Candidate Genes in Faba Bean (Vicia faba L.).

Faba bean (Vicia faba L.) is one of the most widely grown cool season legume crops in the world. Winter faba bean normally has a vernalization requirement, which promotes an earlier flowering and pod setting than unvernalized plants. However, the molecular mechanisms of vernalization in faba bean are largely unknown. Discovering vernalization-related candidate genes is of great importance for faba bean breeding. In this study, the whole transcriptome of faba bean buds was profiled by using next-generation sequencing (NGS) and single-molecule, real-time (SMRT) full-length transcriptome sequencing technology. A total of 29,203 high-quality non-redundant transcripts, 21,098 complete coding sequences (CDS), 1,045 long non-coding RNAs (lncRNAs), and 12,939 simple sequence repeats (SSRs) were identified. Furthermore, 4,044 differentially expressed genes (DEGs) were identified through pairwise comparisons. By Gene Ontology (GO) enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis, these differentially expressed transcripts were found to be enriched in binding and transcription factor activity, electron carrier activity, rhythmic process, and receptor activity. Finally, 50 putative vernalization-related genes that played important roles in the vernalization of faba bean were identified; we also found that the levels of vernalization-responsive transcripts showed significantly higher expression levels in cold-treated buds. The expression of VfSOC1, one of the candidate genes, was sensitive to vernalization. Ectopic expression of VfSOC1 in Arabidopsis brought earlier flowering. In conclusion, the abundant vernalization-related transcripts identified in this study will provide a basis for future researches on the vernalization and faba bean breeding and established a reference full-length transcriptome for future studies on faba bean.

English

Strictosidine synthase, encoded by the gene STR, facilitates the regeneration of strictosidine, a critical intermediate for the synthesis of many plant alkaloids. The gene has, however, never been studied in Cinchona spp. The plants produce quinine alkaloid used for malaria medication, SARS-CoV-2 treatment and other industrial purposes. Cultured cells can produce the alkaloid but only at a much lower yield than the natural tree. This study describes STR expression and quinine content in various plant materials. Bioinformatic analyses were conducted on nine species of Rubiaceae to obtain reference sequences to design conservative primers for Cinchona ledgeriana STR (ClSTR). ClSTR expression was analyzed using qRT-PCR and quinine content was determined using HPLC. A complete coding sequence (CDS) of ClSTR was deposited in NCBI GenBank under the accession number MK422544.1. ClSTR was expressed in cultured cells, young and mature leaves, and stem bark. The elicited cells have higher expression than the control and they performed since the fourth week. However, the quinine content was greater in older cells. The gene expression in young leaves was superior, but quinine was most abundant in the stem bark. Every cell of C. ledgeriana, in culture or in the plant, expressed ClSTR and was capable of synthesizing the alkaloid quinine. The alkaloid from the leaves of the plant might be translocated and accumulated in the bark. No efflux of alkaloids from the confined cultured cells might contribute in triggering feedback inhibition in the biosynthetic pathway. This study revealed a critical obstacle in cell culture as a means of secondary metabolites production that needs further development of metabolic engineering. © 2021 Friends Science Publishers

First report of Turnip yellows virus in cabbage in the Philippines

In March 2017, 52 dried-leaf samples of brassicaceous plants from the Philippine Highlands, one of which was a sample of cabbage (Brassica oleracea var. capitata; Fig. 1), were submitted to Fera Science Ltd., via the Centre for Agriculture and Bioscience International (CABI). The plants were all displaying virus-like, yellowing symptoms (Reeder et al., 2017). Initially, samples were bulked together and tested by ELISA for the presence of viruses known to occur in the Brassicaceae including Cucumber mosaic virus (CMV) (Agdia, USA), Turnip mosaic virus (TuMV) and Turnip yellows virus (TuYV) (Loewe, Germany). All the bulked samples gave negative results for CMV, TuMV and TuYV, and the samples were also tested individually for the presence of Turnip yellow mosaic virus (TYMV) (DSMZ, Germany). All Chinese cabbage (Brassica rapa subsp. pekinensis) samples were positive for TYMV as described by Reeder et al. (2017). However, the Brassica oleracea var. capitata sample tested negative for TYMV and was therefore screened for virus infection using an Illumina MiSeq as described by Adams et al. (2014). A total of 857,018 reads were obtained from the sample, 924 were assembled to yield the complete coding sequence of a polerovirus (GenBank Accession No. MW537050). The average sequencing depth of this contig was 31. Comparison at the whole genome level initially suggested that the isolate was either Brassica yellows virus or TuYV with significant nucleotide identity (>94%) to examples of both viruses. Recombination analysis using RDP 4 (Martin et al., 2015) for both TuYV (NC003743) and BrYV (NC016038) type isolates suggested that this isolate has a small recombination event in the P3-P5 region. Filardo et al. (2021) have proposed that TuYV and BrYV are the same species and describe isolates with a similar P3-P5 recombination as within a clade (group 2) of their unified TuYV species. Therefore, this isolate has been identified as containing a group 2 TuYV. A specific real-time RT-PCR primer/probe set was designed based on both TuYV and BrYV sequences. The primer/probe sequences are as follows TuYV-F2: 5′-GCCGCYTGTTTCTCAGTTCTG-3′; TuYV-R2: 5′-RACTAACCACGAGTAAAGAAGCTCAA-3′; TuYV-P2 (probe): [FAM] 5′-ACGAGTTGCGGCAYGATCCAGC[BHQ1] -3′. Testing the Brassica oleracea var. capitata sample by the real-time RT-PCR assay yielded a positive result, whereas the sample had tested negative by ELISA, suggesting the ELISA does not detect all strains of TuYV. TuYV is widespread in cruciferous plants across mainland China, South Korea and Japan (Zhang et al., 2016) but this is the first report of TuYV in the Philippines. Further work is needed to determine the prevalence of TuYV in cruciferous plants in the Philippines and identify the key aphid vectors. This information could then be used to inform future management strategies. This testing was partially funded through the Defra-Fera Long Term Service Agreement. The authors would like to thank CABI for their support.

Phylogenetic analyses of ribosomal transcription units from Haplorchis taichui and H. pumilio species of Heterophyidae (Platyhelminthes: Opisthorchiata)

Heterophyidiasis caused by minute intestinal flukes becomes of public concern in many countries worldwide. Haplorchis taichui and H. pumilio, belonging to the family Heterophyidae (Platyhelminthes: Trematoda) are two of many infecting humans and commonly found in Vietnam. Sequence study of these two small intestinal flukes is still very limited, hence we need more prospective markers for taxonomic identification and classification. This study provides complete coding sequence of the ribosomal transcription units (rTU) from H. taichui and H. pumilio (Vietnamese samples) and demonstrates the use of complete 28S rDNA sequences for phylogenetic analysis.The complete coding sequence of the rTU (from 5' 18S to 3' 28S), consisting of complete 18S, ITS-1, 5.8S, ITS2 and complete 28S rRNA genes and spacers, from H. taichui (7,268 bp) and H. pumilio (7,416 bp) from human hosts in Vietnam, were determined and annotated. The 18S and 5.8S genes of both species were of the same length (1,992 bp/18S, 160 bp/5.8S), but 28S genes differed (3,875 bp/H. taichui and 3,870 bp/H. pumilio). ITS-1 in H. taichui (797 bp) and ITS-2 in H. pumilio (280 bp) do not contain tandem repeat units (TRUs), while ITS-1 in H. pumilio (1,106 bp) contains 3 TRUs of 136 bp/each and 2 TRUs of 116 bp/each and ITS-2 in H. taichui (444 bp) contain 3 TRUs (83–85 bp/each). A phylogenetic tree inferred from the alignment of complete 28S rDNA sequences of 32 trematode strains/species, including 2 Vietnamese Haplorchis spp. and 24 species of 8 families in the suborders Xiphidiata (families Nanophyetidae, Paragonimidae, Collyriclidae), Opisthorchiata (Heterophyidae, Opisthorchiidae), and Echinostomata (Echinostomatidae, Fasciolidae). and Schistosoma japonicum of the family Schistosomatidae is used as an outgroup. The topology of the phylogenetic tree clearly confirmed the status of the Vietnamese H. taichui and H. pumilio species. These species gathered in a group (in the family Heterophyidae) clearly identified in the position of "sister” group to those in the family Opisthorchiidae (suborder Opisthorchiata, superfamily Opisthorchioidea).

Genetic and evolutionary analysis of emerging HoBi-like pestivirus

HoBi-like pestivirus, an emerging species within the Pestivirus genus, is an important pathogen associated with a variety of clinical manifestations of ruminants, especially cattle. HoBi-like pestiviruses were identified in several countries and from different hosts, and raised concerns with regard to their acute and persistent infections, which is implicated in economic losses for cattle farmers. However, the transmission path, codon usage bias, and host adaptation of the virus has not been studied. Hence, we performed the analysis the spatio-temporal transmission based on the available 5’-UTR sequences of HoBi-like pestivirus, and then conducted codon analysis of the complete coding sequence of the virus. The results show the virus appeared in 1952 (95% HPD: 1905–1985) and may have originated in India. In addition, Italy is the hub for the spread of the virus. Moreover, six potential recombination events and two complex recombination events were discovered. Analysis of codon usage patterns revealed that the effective number of codon (ENC) values with an average of 50.85, and the codon usage bias is greatly affected by natural selection, which is different from the previous BVDV-1, 2. Finally, codon adaptation index (CAI) analysis shows that pigs may be the potential origin species of the HoBi-like pestivirus. These findings will contribute to more effective control of the spread of the virus, extend the knowledge about the genetic and evolutionary features of HoBi-like viruses and provide some information for vaccine research.

RNA in situ hybridization and expression of related genes regulating the accumulation of triterpenoids in Cyclocarya paliurus.

Cyclocarya paliurus (Batal.) Iljinskaja, a woody medicinal species in the Juglandaceae, grows extensively in subtropical areas of China. Triterpenoids in the leaves have health-promoting effects, including hypoglycemic and hypolipidemic activities. To understand triterpenoid biosynthesis, transport and accumulation in C. paliurus during the growing season, gene cloning, gene expression and RNA in situ hybridization of related genes were used, and accumulation was examined in various organs. The complete coding sequences (CDSs) of three genes, CpHMGR, CpDXR and CpSQS, were obtained from GenBank and RACE. RNA in situ hybridization signals of the three genes mainly occurred in the epidermis, palisade tissue, phloem and xylem of leaf, shoot and root, with the signals generally consistent with the accumulation of metabolites in tissues, except in the xylem. Both gene expression and triterpenoid accumulations showed seasonal variations in all organs. However, total triterpenoid content in the leaves was significantly higher than that in the shoots, with the maximum in shoots in August and in leaves in October. According to Pearson correlation analysis, triterpenoid accumulation in the leaves was significantly positively related with the relative expression of CpSQS. However, the relation between gene expression and accumulation was dependent on the role of the gene in the pathway as well as on the plant organ. The results suggested that most of the intermediates catalyzed by CpHMGR and CpDXR in young shoots and roots were used in growth and flowering in the spring, whereas subsequent triterpenoid biosynthesis in the downstream catalyzed by CpSQS mainly occurred in the leaves by using transferred and in situ intermediates as substrates. Thus, this study provides a reference to improve triterpenoid accumulation in future C. paliurus plantations.

Genome Sequence of a Yunnan Orbivirus Isolated from a Dead Florida White-Tailed Deer (Odocoileus virginianus).

We report the complete coding sequences of a Yunnan orbivirus isolated from a dead white-tailed deer (Odocoileus virginianus) in Florida in 2019. The prevalence of Yunnan orbivirus and its role in disease among farmed white-tailed deer remain to be determined.

Identification and molecular profiling of a novel homolog of cystatin C from rock bream (Oplegnathus fasciatus) evidencing its transcriptional sensitivity to pathogen infections.

Cystatins are reversible inhibitors of cysteine proteases which show an omnipresent distribution in the life on earth. Although, cystatins with mammalian origin were well characterized and their roles in physiology were reported in details, those from teleostean origin are still underrepresented in literature. However, role of cystatins in fish physiology and immune defense is highlighted in few recent reports. In this study, a cystatin C holmologue from rock bream (Oplegnathus fasciatus); termed RbCytC was identified and molecularly characterized. The complete coding sequence of RbCytC was 387bp in length, which codes for a polypeptide with 129 amino acids, including a signal peptide of 19 amino acids. The consensus cystatin family signatures including a G residue, turning up towards the N-terminus region, QVVAG motif, locating at the middle of the sequence and the PW motif at the c terminal region was found to be well conserved in RbCytC. Phylogenetic analysis using different cystatin counterparts affirmed the close evolutionary relationship of RbCytC with its teleostan homologs which belong to family 2 cystatins. The predicted molecular model of RbCytC resembled most of the structural features of empirically elucidated tertiary structures for chicken egg white cystatin. According to the qPCR assays, RbCytC showed detectable expression in all fish tissues used in the experiment, with markedly pronounced expression level in liver. Moreover, its basal mRNA expression was up-regulated in liver and spleen tissues by experimental rock bream iridovirus infection, whereas down regulated in the same tissues, post live Edwardsiella tarda injection. Collectively, outcomes of our study validate the structural homology of RbCytC with known cystatin C similitudes, especially those of teleosts and suggest its potential roles in proteolytic processes of rock bream physiology as well as in host immune defense mechanisms.