Nucleotide Sequence Homology Research Articles

Molecular Characteristics of Indonesian Isolate <i>Enterocytozoon hepatopenaei</i> Based on Sequence Analysis of 18S rRNA Genes

ABSTRACT Enterocytozoon hepatopenaei (EHP) infection have been reported as an obstacle of whiteleg shrimp (Penaeus vannamei) culture in Indonesia. However, understanding of the molecular characteristics of EHP species in Indonesia is not widely known. The aims of this study were to determine the identity and characters of DNA, and their phylogeny of EHP species from several different locations in Indonesia with specific references to 18S rRNA gene. The EHPs were collected from cultured P.vannamei from Lampung, Pangandaran, Sidoarjo, Banyuwangi, Probolinggo, Blitar, Makassar, and Lombok. Thirteen (13) samples were randomly selected to explore their gene characters through 18S rRNA gene sequencing. The primers used were EHP_F and EHP_1R. Parameter observed were DNA sequencing, nucleotide sequence homology with related available genes in the Gen Bank database, multiple sequence alignment, and reconstruction of genetic relationship trees. DNA sequence homology analysis showed that all samples had 99.89-100% similarity to Indian Enterocytozoon hepatopenaei (Accession Number MH259890.1 and MH260592.1). The alignment results illustrated that all EHP sequences of Indonesian isolates were 100% identical each other. The phylogenetic tree topology provided information that all sample accessions were in the same clade and spread evenly. The conclusion were that the Indonesian EHP species were identical (100%) and it could be said that they were genetically homogeneous. Keywords: Enterocytozoon hepatopenaei, 18S rRNA gene, Indonesia.

Identification of the bacterium isolate from Mackerel Fish (Rastrelliger sp.) using 16S rRNA Gene

The use of probiotics in aquaculture can improve the quality and quantity of the fishes. One of potential probiotic bacterium from gastrointestine of mackerel fish (Rastrelliger sp.) isolated in this research were LUI-04. This study aimed to determine the species of bacterium isolate using nucleotide sequence homology compared to other microorganisms in GenBank. The 16S rRNA genes of LUI-04 isolate were analyzed. The results of electrophoresis showed the DNA band had a size of 1 500 bp for amplification using Bact-27F and Uni-1492R primers, and about 250 bp to 500 bp with the Com-IF and Com-2R primers. The sequence of nucleotides was determined by Sanger method. The 16S rRNA sequence of the isolate and other bacteria in GenBank were compared with the Basic Alignment Search Tool (BLAST) to know the homology. Then, all of the sequences were aligned by ClustalW, and the phylogenetic tree was reconstructed with Neighbor Joining method using bootstrap analysis of 1 000 replicates. The results showed that LUI-04 had a homology of 99 % and the bootstrap value percentage of 86 % with Bacillus aryabhattai B8W22 and four strains of Bacillus megaterium (strain QM B1551, NBRC15308, strain IAM 13418 and ATCC 14581). Therefore, the LUI-04 isolate belongs to Bacillus megaterium species.

Nonsense mutations and genetic compensation response

The genetic compensation response (GCR) was firstly described in zebrafish to explain the phenotypic discrepancies between gene-knockout and gene-knockdown, whereby a deleterious mutation, but not gene-knockdown, can lead to the transcriptional upregulation of related genes, which can assume the function of the mutated gene. This phenomenon was also found in other model systems including mice and Arabidopsis. However, the underlying molecular mechanism of the GCR remains elusive until two papers were published in Nature on April 3, 2019: one from our lab and the other from Stainier's lab. Using different genetic mutants of various genes in zebrafish or culture cells of mice, both of us reveal that the upregulation of compensatory genes is only triggered by mutations that generate a premature termination codon (PTC); the compensatory genes share nucleotide sequence homology to the mutated genes; nonsense mRNA mediated decay pathway (NMD) is essential for the induction of GCR, and the increased transcription of the compensatory genes is accompanied by an enhancement of H3K4 trimethylation (H3K4me3) at their transcription start site (TSS) regions. In this review, we summarize the mechanisms of the GCR proposed in the two studies.

A Cluster of Bunyavirus-Associated Severe Fever With Thrombocytopenia Syndrome Cases in a Coastal Plain Area in China, 2015: Identification of a Previously Unidentified Endemic Region for Severe Fever With Thrombocytopenia Bunyavirus.

BackgroundSevere fever with thrombocytopenia syndrome (SFTS) is a typical tick-borne, natural focal disease. The natural foci of SFTS were considered to exist in hilly and mountainous areas before 2015. A cluster of 3 patients exposed to a patient with a fulminant disease consistent with SFTS occurred from July to August 2015 in Dongtai County, which is characterized by alluvial plains; this prompted investigation.MethodsThe epidemiological, clinical, and laboratory features of 4 patients in the cluster were analyzed. Serum samples from the indigenous healthy population and native domesticated animals were collected to conduct laboratory tests, along with small wild animals and ticks.ResultsIn 3 secondary case patients, high fever, thrombocytopenia and leukopenia developed within 8–13 days after contact with blood or bloody secretions from the index patient; SFTS was then diagnosed by means of reverse-transcription polymerase chain reaction. Genomic sequencing and analysis of S and L segments of 2 viral strains isolated from 2 secondary case patients showed that they shared 99.8%–99.9% homology in nucleotide sequence. The seroprevalences among indigenous healthy population, native livestock, native poultry, and small wild animals was 0.74%, 17.54%, 6.67%, and 1.12%, respectively. Three questing ticks, 61 feeding ticks, and 178 small wild animals were collected in August 2015. Survey on tick density and seasonal fluctuation in 2016 showed that ticks were active from March to October. All ticks were identified as Haemaphysalis longicornis. Severe fever with thrombocytopenia bunyavirus (SFTSV)–specific RNA was detected in the ticks collected in 2016, and the minimum SFTSV infection rate in these ticks was 0.54% (1 of 185).Wild mammals and ticks collected in August 2015 tested negative for SFTSV-specific RNA.ConclusionsAside from hilly or mountainous area, a coastal plain was identified as the natural foci of SFTSV in Dongtai County, China. The involvement of migration in the evolution of SFTSV might lead to a transregional transmission event of SFTSV.

Isolation and identification of Singapore grouper iridovirus Hainan strain (SGIV-HN) in China.

In recent years, with the rapid development of marine farming activities, outbreaks of viral diseases have affected the grouper aquaculture industry, causing heavy economic losses. Singapore grouper iridovirus (SGIV) is one of the most important viruses causing disease in fish. In the present study, we isolated and identified a virus from diseased groupers by coculturing the affected tissue cells with grouper spleen cells. The genome of the isolated virus shared 99.83% nucleotide sequence homology with those of SGIV reference strains in the GenBank database. The virus clustered with SGIV on an evolutionary tree constructed based on "major capsid protein" (MCP) amino acid sequences, so it was designated 'Singapore grouper iridovirus Hainan' (SGIV-HN). To evaluate the pathogenic potential of SGIV-HN in fish, orange-spotted groupers were infected by intraperitoneal injection with the virus. Infected groupers began to die from the fourth day after infection, and survivors tended to be stable by the eighth day. The death rate was 83.33%. In a mock-infected control group, only two fish died, and the mortality rate was 6.67%. Dissection showed that the fish had enlarged spleenswith hemorrhage, and enlarged cells were visible with Giemsa staining. This is the first report of isolation of SGIV from naturally infected fish in China, and we show that SGIV-HN is highly infectious, causing massive deaths in groupers.

Molecular characterization and phylogenetic analysis of tomato leaf curl Palampur virus, a bipartite begomovirus, associated with Cucumis sativus L. in Pakistan.

Leaf samples of Cucumis Sativus L. (C. sativus) (Family; Cucurbitaceae) showing vein thickening, mild leaf curling and leaf enations were collected from the farmer's field. Amplification of the full-length viral molecules was performed through rolling circle amplification (RCA). Cloning of the full-length viral molecules was done through standard cloning procedure followed by sequencing. Sequence similarity analysis and phylogenetic studies showed that the virus associated with leaf curling and enations in C. sativus was a bipartite begomovirus, where DNA-A and DNA-B showed highest nucleotide sequence homology of 98% and 97% to tomato leaf curl Palampur virus (ToLCPMV) from India. Attempts to isolate betasatellites and alphasatellites through PCR using RCA product as template, did not result in any amplification. A maximum likelihood phylogenetic tree grouped DNA-A and B components with other isolates from India. SDT was used to find the pairwise identity scores of different sequences of ToLCPMV present in the database. Phylogenetic analysis showed that sequences of ToLCPMV DNA-A and B components in this study share high degree of homology with existing viruses and are isolates of ToLCPMV-India. Infectious molecules of both components (Accessions, MG252783 and MG252784, respectively) were constructed for infectivity analysis to fulfill the Koch's postulate. Infectivity analysis revealed that ToLCPMV DNA-A is infectious to model host plant Nicotiana benthamiana and viral accumulation was confirmed through Southern blot analysis. Accumulation of DNA-B was confirmed through PCR. Infectivity analysis was also conducted using the original host, C. sativus, but plants were unable to survive the agroinoculation. To our knowledge this is the first report of ToLCPMV associated with C. sativus L. in Pakistan.

Characterization of one new non-S-RNase of Armeniaca cathayana

Armeniaca cathayana, a new species described in 2010, belongs to gametophytic self-incompatibility (GSI) system which is under S-allele control. One new non-S-ribonuclease (non-S-RNase) was found in A. cathayana through comparing its nucleotide and amino acid sequences with sequences of the S-allele in Genbank. The BLAST analysis showed that the one new non-S-RNase S68-RNase (GenBank Accession No. MH155952) had the highest 96% nucleotide sequence homology with Prunus webbii non-S-RNase PW<sub>1</sub> (EU809938.1). Alignment of deduced amino acid sequences of A. cathayana S68-RNase shared 83% similarity with P. webbii PW<sub>1</sub>. The new non-S-RNase determined in this study will provide new information to GSI of Rosaceae.

Mycobacteriophage CRB2 defines a new subcluster in mycobacteriophage classification.

Mycobacteriophages are viruses -mostly temperates- that infect Mycobacterium smegmatis and sometimes Mycobacterium tuberculosis. Mycobacteriophages are grouped in clusters on the basis of the overall nucleotide sequence homology, being further divided in subclusters as more mycobacteriophage genomes are sequenced and annotated. As part of our on-going screening for novel isolates, we herein report the bioinformatics analysis of CRB2, a mycobacteriophage belonging into the Siphoviridae family that propagates at 30°C. CRB2 has a 72,217 bp genome with a 69.78% GC content that belongs to Cluster B; nucleotide comparison with other B cluster members positions CRB2 as the sole member of a new subcluster, B9, being mycobacteriophage Saguaro (belonging into subcluster B7) its closest relative. Sequencing and annotation of 14 mycobacteriophages isolated by our group has yielded six cluster A members, a singleton, four of the five members of subcluster B6, one of the three reported members of subcluster G4, and CRB2 which defines subcluster B9. Considering the massive mycobacteriophage search performed in USA and the relatively rarity of our phages, we propose that factors other than size of the sampling determine the variability of mycobacteriophage distribution, and thus a world-wide concerted mining would most likely bring extremely rare and yet undiscovered mycobacteriophages.

Sequence analysis of VP1-VP4 genes of coxsackievirus A6 strains isolated from children with severe hand, foot, and mouth disease in Shenzhen from 2012 to 2015

Objective To analyze the genetic characteristics of VP1-VP4 genes carried by coxsackievirus A6 (CVA6) strains isolated from severe cases of hand, foot, and mouth disease (HFMD) in Shenzhen during 2012 to 2015. Methods The VP1-VP4 genes of CVA6 strains isolated from severe HFMD cases in Shenzhen during 2012 to 2015 were amplified and sequenced. Phylogenetic analysis was performed to analyze the VP1-VP4 genes of CVA6 isolates and sequences downloaded from GenBank by using DNASTAR6.0 and MEGA6.02 software packages. Results Four cases of severe HFMD were caused by CVA6 in Shenzhen during 2012 to 2015. All of the patients had the symptom of fever, skin rash and aseptic encephalitis. The CVA6 strain causing severe HFMD in 2013 shared 98.8%-98.9% homology in nucleotide sequences and 99.3%-99.8% in amino acid sequences with the strains isolated in 2012. Two amino acid mutations were found in the CVA6 strain isolated in 2013, which were G73E in VP2 region and S13G in VP1 region. However, the CVA6 strain isolated in 2015 only shared 95.0% homology in nucleotide sequences and 99.3% homology in amino acid sequences with the strain isolated in 2013. Six amino acid mutations were identified including E73G in VP2 region and T5A, S27N, A30V, N137S and V242I in VP1 region. The phylogenetic analysis revealed that the four CVA6 strains belong to D3 sub-genotype. The CVA6 strains causing severe cases in 2012 had the nearest genetic relationship with the strain isolated in Changsha in 2012 (KJ156349). The CVA6 strain isolated in Shenzhen in 2013 had the nearest genetic relationship with the strain isolated in Shanghai in 2013 (KJ612513). The Shenzhen CVA6 isolate in 2015 showed high similarity to Weifang CVA6 isolate in 2014 (KX752785). Conclusions All CVA6 strains causing severe HFMD cases in Shenzhen during 2012 to 2015 belongs to D3 sub-genotype. Mutations of S27N and A30V in the VP1 region of the CVA6 isolate in 2015 are located in the B cell epitopes. In addition, the VP1-V242I mutation in the CVA6 strain isolated in 2015 is located in the binding site of PSGL-1 receptor. These mutations may affect the binding of CVA6 strains to the cellular receptors and their infectivity to people. Key words: Hand, foot, and mouth disease; Coxsackievirus A6; Severe case; Genotype; Mutation

Comparative analysis of the complete chloroplast genome sequences of three Amaranthus species

AbstractThe complete chloroplast (cp) genome sequences of threeAmaranthusspecies (Amaranthus hypochondriacus, A. cruentusandA. caudatus) were determined by next-generation sequencing. The cp genome sequences ofA. hypochondriacus,A. cruentusandA. caudatuswere 150,523, 150,757 and 150,523 bp in length, respectively, each containing 84 genes with identical contents and orders. Expansion or contraction of the inverted repeat region was not observed among the threeAmaranthusspecies. The coding regions were highly conserved with 99.3% homology in nucleotide and amino acid sequences. Five genes –matK,accD,ndhJ,ccsAandndhF –showed relatively high non-synonymous/synonymous values (Ka/Ks> 0.1). Sequence comparison identified two insertion/deletion (InDels) greater than 40 bp in length, and polymerase chain reaction markers that could amplify these InDel regions were applied to diverse Korean Genbank accessions, which could discriminate the threeAmaranthusspecies. Phylogenetic analyses based on 62 protein-coding genes showed that the core Caryophyllales were monophyletic and Amaranthoideae formed a sister group with the Betoideae and Chenopodioideae clade. Comparing each homologous locus among the threeAmaranthusspecies, identified eight regions with high Pi values (>0.03). Seven of these loci, except forrps19-trnH(GUG), were considered to be useful molecular markers for further phylogenetic studies.

Seromonitoring of Peste des Petits Ruminants in goats and molecular characterization of PPR virus from field cases.

Objectives:The study was undertaken with the objectives to perform seromonitoring of Peste des Petits Ruminants (PPR) antibodies in goats vaccinated with PPR vaccine and molecular characterization of PPR virus (PPRV) from field cases in Bangladesh.Materials and Methods:Seromonitoring work was conducted in Char Kalibari, Mymensingh Sadar, Mymensingh. For this, a total of 50 goats were randomly selected and were divided into two groups; vaccinated (Group A; n = 25) and non-vaccinated (Group B; n = 25). The goats of both groups were again sub-divided into four age groups; (i) 0–6 months (n = 5), (ii) 6–12 months (n = 5), (iii) 12–24 months (n = 10), and (iv) >24 months (n = 5). Blood samples were collected on Day-0 and after 21 days of post-vaccination (DPV), and the sera were prepared. The sera were examined for the presence of antibodies against PPRV by competitive enzyme-linked immunosorbent assay. For molecular characterization, nasal swabs (n = 10) were collected from PPR infected goats in Jessore during PPR outbreak (February 2016). The causative agent, PPRV isolated from field cases were confirmed by N gene based on reverse transcription polymerase chain reaction (RT-PCR), followed by sequencing, phylogenetic analysis, and multiple sequence alignment analyses.Results:In the case of seromonitoring, the results revealed that before vaccination (at Day-0), overall, 44% (n = 22/50) goats were seropositive for PPRV. In Group A, 48% (n = 12/25) goats were seropositive, but after 21 DPV, 96% (n = 24/25) goats become seropositive. On the other hand, in Group B, 40% (n = 10/25) and 16% (n = 04/25) seropositive goats found at Day-0 and after 21 DPV, respectively, indicating that the antibody titer was increasing after vaccination and decreasing in convalescent goats. Out of 10 nasal swab samples, 40% (n = 4/10) was confirmed by RT-PCR targeting nucleocapsid (N gene). Phylogenetically, our isolate (KY039156/PPRV/BDG/Jes/2016) was similar to the other strains of PPRV under lineage IV. However, there was a unique amino acid substitution, where glycine (G) was recorded in place of arginine (R). The strain is closely related with other Chinese or Indian strains. The nucleotide sequence homology by NCBI BLAST search of the isolated strain ranged from 95% to 99% with other strains circulating in Bangladesh.Conclusion:The PPRV is prevailing in the Mymensingh and Jessore regions of Bangladesh. Effective control of PPR in goats may depend on vaccination with PPR vaccine. Molecular characterization of PPRV in Jessore reveals that the virus is differing from the strain prevalent in other regions of Bangladesh and the world.

The pervasive effects of recombinant Fasciola gigantica Ras-related protein Rab10 on the functions of goat peripheral blood mononuclear cells

BackgroundFasciola gigantica-induced immunomodulation is a major hurdle faced by the host for controlling infection. Here, we elucidated the role of F. gigantica Ras-related protein Rab10 (FgRab10) in the modulation of key functions of peripheral blood mononuclear cells (PBMCs) of goats.MethodsWe cloned and expressed recombinant FgRab10 (rFgRab10) protein and examined its effects on several functions of goat PBMCs. Protein interactors of rFgRab10 were predicted in silico by querying the databases Intact, String, BioPlex and BioGrid. In addition, a total energy analysis of each of the identified interactions was also conducted. Gene Ontology (GO) enrichment analysis was carried out using FuncAssociate 3.0.ResultsThe FgRab10 gene (618 bp), encodes 205-amino-acid residues with a molecular mass of ~23 kDa, had complete nucleotide sequence homology with F. hepatica Ras family protein gene (PIS87503.1). The rFgRab10 protein specifically cross-reacted with anti-Fasciola antibodies as shown by Western blot and immunofluorescence analysis. This protein exhibited multiple effects on goat PBMCs, including increased production of cytokines [interleukin-2 (IL-2), IL-4, IL-10, transforming growth factor beta (TGF-β) and interferon gamma (IFN-γ)] and total nitric oxide (NO), enhancing apoptosis and migration of PBMCs, and promoting the phagocytic ability of monocytes. However, it significantly inhibited cell proliferation. Homology modelling revealed 63% identity between rFgRab10 and human Rab10 protein (Uniprot ID: P61026). Protein interaction network analysis revealed more stabilizing interactions between Rab proteins geranylgeranyltransferase component A 1 (CHM) and Rab proteins geranylgeranyltransferase component A 2 (CHML) and rFgRab10 protein. Gene Ontology analysis identified RabGTPase mediated signaling as the most represented pathway.ConclusionsrFgRab10 protein exerts profound influences on various functions of goat PBMCs. This finding may help explain why F. gigantica is capable of provoking recognition by host immune cells, less capable of destroying this successful parasite.

Phylogenetic analysis of the cytochrome P450 (CYP450) nucleotide sequences of the horse and predicted CYP450s of the white rhinoceros (Ceratotherium simum) and other mammalian species

BackgroundThe plight of the white rhinoceros (Ceratotherium simum) and the increasing need of treatment options for injured poaching victims led to the necessity to expand the knowledge on applicable drugs in this endangered species. With very little information available on drug pharmacokinetics in rhino, veterinarians have to rely on information generated from other species. The horse being a closely related species, has served as the model for dose extrapolations. However, from recent research on enrofloxacin and carprofen, the white rhino showed considerable differences in the pharmacokinetic properties of these drugs in comparison to the horse. While the reason for the differences is unknown, a likely cause may be a difference in present cytochrome P450 (CYP450), which may result in the rhino being genetically deficient in certain enzyme families.MethodsFor this paper we assess the degree of similarity of the CYP genome sequences across the different species, using BLAT (BLAST-like alignment tool) for the alignment of the nucleotide sequences of the equine CYP450 with potential homologous nucleotide sequences of the published database from white rhinos and other mammalian species (cow, pig, dog, sheep, elephant, mouse and human).ResultsThe white rhino nucleotide sequences were 90.74% identical to the equine sequences. This was higher than the degree of similarity between any of the other evaluated species sequences. While no specific CYP family were found to be deficient in the published rhino genome, the horse genome contained additional genetic sequence for a larger number of iso-enzymes that were not present in the rhino.DiscussionIn pharmacokinetic study, it is well known that absence of a metabolic enzyme will result in constraints in drug metabolism and drug elimination. While this was our speculation, comparison to the horse and other mammalian species indicate that all the described CYP genes required for metabolism are present within the rhino genome. These results leave functional differences in enzyme activity and a lack of isoenzymes as the likely reason for the constraint in drug metabolism. Despite a more than 90% similarity of the equine and rhino gene sequences, seemingly small differences can have major effects on drug metabolism. Thus, in spite of the close anatomical relationship, the rhino should not simply be treated like a big horse.

CUDAMPF++: A Proactive Resource Exhaustion Scheme for Accelerating Homologous Sequence Search on CUDA-Enabled GPU

Genomic sequence alignment is an important research topic in bioinformatics and continues to attract significant efforts. As genomic data grow exponentially, however, most of alignment methods face challenges due to their huge computational costs. HMMER, a suite of bioinformatics tools, is widely used for the analysis of homologous protein and nucleotide sequences with high sensitivity, based on profile hidden Markov models (HMMs). Its latest version, HMMER3, introdues a heuristic pipeline to accelerate the alignment process, which is carried out on central processing units (CPUs) with the support of streaming SIMD extensions (SSE) instructions. Few acceleration results have since been reported based on HMMER3. In this paper, we propose a five-tiered parallel framework, CUDAMPF++, to accelerate the most computationally intensive stages of HMMER3's pipeline, multiple/single segment Viterbi (MSV/SSV), on a single graphics processing unit (GPU). As an architecture-aware design, the proposed framework aims to fully utilize hardware resources via exploiting finer-grained parallelism (multi-sequence alignment) compared with its predecessor (CUDAMPF). In addition, we propose a novel method that proactively sacrifices L1 Cache Hit Ratio (CHR) to get improved performance and scalability in return. A comprehensive evaluation shows that the proposed framework outperfroms all existig work and exhibits good consistency in performance regardless of the variation of query models or protein sequence datasets. For MSV (SSV) kernels, the peak performance of the CUDAMPF++ is 283.9 (471.7) GCUPS on a single K40 GPU, and impressive speedups ranging from 1.x (1.7x) to 168.3x (160.7x) are achieved over the CPU-based implementation (16 cores, 32 threads).

A new Tanay virus isolated from mosquitoes in Guangxi, China.

In 2013, seven virus strains were isolated from Culex tritaeniorhynchus and C. quinquefasciatus collected in Guangxi Province, China. The viruses caused cytopathological effects (CPE) only in C6/36 cells, and not in mammaliancells, e.g. BHK21 and Vero cells. Comparative genomic analysis revealed that the genomes of the seven virus strains ranged from 9589 to 9595 nt with G+C contents of 37.51-37.59%, and nucleotide sequence similarity > 99.8%. The nucleotide sequence homology of the seven virus strains with a Tanay virus isolate was 79.8-80.0%, while the corresponding amino acid homology of ORF1, ORF2, and ORF3 was 79.8-80, 73.9-4.6, and 93.4-94.3%, respectively. In phylogenetic analysis, based on the RdRP amino acid sequence, the seven virus strains were located on the same evolutionary branch, yet distinct from Tanay virus isolated from the Philippines. These results indicated that the seven virus strains isolated from Guangxi represent a new Tanay virus, "Tanay-like virus", within the genus Sandewavirus of the newly proposed taxon Negevirus. This was the first isolation of Tanay virus in China.

First Report of Maize Yellow Mosaic Virus on Zea mays in South Korea.

Maize yellow mosaic virus (MaYMV) is a novel putative polerovirus recently found in maize (Zea mays) in China and has been detected not only in Asia but also in Africa and South America (Chen et al. 2016; Goncalves et al. 2017; Palanga et al. 2017). In addition, MaYMV infection has also been reported in sugarcane (Saccharum spp.) and itch grass (Rottboellia cochinchinensis) (Yahaya et al. 2017). Recently, the first occurrence of MaYMV in South Korea has been reported from Panicum miliaceum and Sorghum bicolor using high-throughput RNA sequencing (Lim et al. 2018). Therefore, to confirm whether MaYMV exists in maize in South Korea, a total of 26 maize samples with yellowing, yellow stripe, and chlorosis symptoms were collected from three provinces (Chungcheongbuk-do, Gangwon-do, and Gyeongsangnam-do) in August and September 2017. Total RNA was extracted from all samples using the easy-spin Total RNA Extraction Kit (iNtRON Biotechnology, Seongnam, Korea). Reverse transcription polymerase chain reaction (RT-PCR) was conducted using SuPrimeScript RT-PCR Premix (GeNet Bio, Daejeon, Korea) and MaYMV-specific primers (Chen et al. 2016), producing an amplicon with expected size of 753 bp, using protocols described previously (Lim et al. 2018). Five out of six samples collected from Chungcheongbuk-do, five out of 10 samples collected from Gangwon-do, and one out of 10 samples collected from Gyeongsangnam-do were confirmed to be infected with MaYMV. One RT-PCR fragment of MaYMV from each region (Gangwon-do, Chungcheongbuk-do, and Gyeongsangnam-do) was directly sequenced (Macrogen, Daejeon, Korea), and the sequences were deposited in GenBank under accession numbers MG020517, MG020518, and MG020519, respectively. The partial nucleotide sequences of MaYMV determined in each sample collected from Chungcheongbuk-do and Gyeongsangnam-do were identical, and these nucleotide sequences differed by one nucleotide from that identified in the sample collected from Gangwon-do. The NCBI BLAST search showed that the three MaYMV isolates shared high nucleotide sequence homology (99 to 100%) with isolates Yunnan3 (KU291099) and Yunnan4 (KU291100) from maize in China. The MaYMV-infected samples showed yellow stripe and chlorosis symptoms, which tend to first appear on the tip and edge of the leaves and become worse. However, further studies are needed, because it is uncertain whether the symptoms were caused by a single infection with MaYMV. To the best of our knowledge, this is the first report of MaYMV on Z. mays in South Korea.

Prevalence and molecular subtyping of Blastocystis from dairy cattle in Kanagawa, Japan

Blastocystis is an intestinal protist, commonly found in the human population and in a wide range of animals globally. Currently, isolates from mammalian and avian hosts are classified into 17 subtypes (STs) based on phylogeny of the small subunit rRNA gene (SSU rDNA), of which ten (ST1-9, 12) are reported in humans. ST10 is a major ST reported from livestock cattle. However, other STs including ST1, 3, 4, 5, and 6, which have the potential to be transmitted to humans, are also reported from cattle in several countries. Although a survey has been conducted previously in western Japan for livestock cattle, there is no information available regarding other parts of Japan. Therefore, this study surveyed the prevalence of Blastocystis and its STs in cattle from Kanagawa prefecture, eastern Japan. Fecal specimens, collected from 133 dairy cattle on four different farms, were subjected to a short-term xenic in vitro culture and Blastocystis were identified by microscopic examination. Seventy-two cattle were positive for Blastocystis (54.1%). Direct sequences for the partial SSU rDNA were obtained for 45 samples. Based on nucleotide sequence homology search and phylogenetic analysis, 44 isolates were identified as ST14 and one as ST10. Our study confirms the presence of these STs in dairy cattle in Japan for the first time. The STs identified here, ST10 and ST14, support previous findings that Bovidae may be the natural host for both STs.

Differential expression and evolution of three tandem, interleukin-1 receptor-like 1 genes in rainbow trout (Oncorhynchus mykiss)

Interleukin-1 receptor-like 1 (Il1rl1 or ST2), a member of the interleukin-1 receptor family, has pleiotropic roles including tissue homeostasis, inflammation, immune polarization, and disease resistance in mammals. A single orthologue was previously described in salmonid fish; however, a recently improved genome assembly of rainbow trout (Oncorhynchus mykiss) revealed three adjacent, tandem il1rl1 orthologues on chromosome Omy 03. Here, we report the genomic organization and evolution of the three il1rl1 genes (il1rl1α, il1rl1β, il1rl1γ), and use both RNA-seq and gene-specific qPCR methods to quantify expression patterns. Nucleotide sequence homology between the three genes is >95% and each predicted protein contains three IG/IG-like domains, a transmembrane region and a TIR domain. The amino acid sequence homology of the rainbow trout il1rl1 genes are highly related to two functional copies in Atlantic and Coho salmon (∼94%) but relatively low (22–26%) with avian and mammalian species. Transcript abundance measured by RNA-seq in 15 tissues of healthy adult rainbow trout indicate constitutive expression of each gene. In whole body lysates, il1rl1α was shown to have >20 fold mRNA expression compared to il1rl1β and il1rl1γ as measured by qPCR assays specific to il1rl1α or il1rl1γ, as well as a multi-gene qPCR assay (il1rl1α,β,γ). Unrooted phylogenetic trees grouped the rainbow trout il1rl1 genes apart from other interleukin-1 receptor family genes and genomic comparisons identify preserved synteny between mammals, birds and salmonids albeit a pseudogene is present in both Atlantic salmon and Coho salmon. Phylogenetic analyses suggest that the three genes arose by tandem duplication but are inconclusive whether these events occurred prior-to or after salmonid speciation. These findings further the understanding of interleukin receptor family evolution and their contribution to teleost immune function.

Reproductive Long Intergenic Noncoding RNAs Exhibit Male Gamete Specificity and Polycomb Repressive Complex 2-Mediated Repression.

Long noncoding RNAs (lncRNAs) have been characterized extensively in animals and are involved in several processes, including homeobox gene expression and X-chromosome inactivation. In comparison, there has been much less detailed characterization of plant lncRNAs, and the number of distinct lncRNAs encoded in plant genomes and their regulation by developmental and epigenetic mechanisms remain largely unknown. Here, we analyzed transcriptome data from Asian rice (Oryza sativa) and identified 6,309 long intergenic noncoding RNAs (lincRNAs), focusing on their expression in reproductive tissues and organs. Most O. sativa lincRNAs were expressed in a highly tissue-specific manner, with an unexpectedly high fraction specifically expressed in male gametes. Mutation of a component of the Polycomb Repressive Complex2 (PRC2) resulted in derepression of another large class of lincRNAs, whose expression is correlated with H3K27 trimethylation in developing panicles. Overlap with the sperm cell-specific lincRNAs suggests that epigenetic repression of lincRNAs in the panicles was partially relieved in the male germline. Expression of a subset of lincRNAs also showed modulation by drought in reproductive tissues. Comparison with other cereal genomes showed that the lincRNAs generally have low levels of conservation at both the sequence and structural levels. Use of a novelty detection support vector machine model enabled the detection of nucleotide sequence and structural homology in ∼10% and ∼4% of the lincRNAs in genomes of purple false brome (Brachypodium distachyon) and maize (Zea mays), respectively. This is the first study to report on a large number of lncRNAs that are targets of repression by PRC2 rather than mediating regulation via PRC2. That the vast majority of the lincRNAs reported here do not overlap with those of other rice studies indicates that these are a significant addition to the known lincRNAs in rice.

Deep sequencing analysis of a strain of pecan mosaic-associated virus infecting Atractylodes macrocephala Koidz

Leaves of Atractylodes macrocephala Koidz with virus-like symptoms were collected in Shanxi Province, China. The strain Am of the Potyvirus species, pecan mosaic-associated virus (PMaV-Am), was discovered through the assembly of virus derived small interfering RNAs (vsiRNAs) that were isolated from symptomatic samples. The complete genome of PMaV-Am was determined to be 9310 nucleotides (nt) in length, including a large open reading frame encoding a polyprotein. Phylogenetic analysis showed that PMaV-Am is clustered together and most closely related to strain PMaV-LA from pecan. Sequence alignments of PMaV-Am complete genomic RNAs with PMaV-LA showed 98.78% nucleotide sequence homology and 96.53% amino acid identity, respectively. Further analyses of the vsiRNA profile showed that the most abundant PMaV from A. macrocephala (PMaV-Am) vsiRNAs were 21 nt long and biased for A at their 5′ terminal residue. Polarity distribution revealed that vsiRNAs were nearly equal amount of sense and antisense strands and hotspot was distributed in the HC-Pro coding region of the PMaV-Am genome. These results might provide an insight into the host RNA silencing defense induced by PMaV-Am, and provide guidelines on designing antiviral strategies using RNAi against PMaV-Am.