Complete genome analysis of enterotoxigenic Escherichia coli K88 phage vB_EcoP_E212, a novel genus Lederbergvirus

The features and genome annotation of a newly bacteriophage v B_EcoP_E212 (referred to as E212) which isolated from farm sewage collected in Jilin, China was describes in this study. Bacteriophage E212 belongs to the family Podoviridae, order Caudovirales through transmission electron microscopy. This phage specifically infects enterotoxigenic Escherichia coli K88. The dsDNA molecule of phage E212 was 38252 bp in length and contained 46.98% G + C content. It has been predicted to contain 53 ORFs, and no tRNAs. Phage E212 carried the integrase gene, and no homologues of virulence factors or antimicrobial resistance genes were found in this phage. Phage E212 was assigned to the genus Lederbergvirus in accordance with nucleotide sequence alignment and phylogenetic analysis.

ChIP-seq Analysis of the Global Regulator Vfr Reveals Novel Insights Into the Biocontrol Agent Pseudomonas protegens FD6

Many Pseudomonas protegens strains produce the antibiotics pyoluteorin (PLT) and 2,4-diacetylphloroglucinol (2,4-DAPG), both of which have antimicrobial properties. The biosynthesis of these metabolites is typically controlled by multiple regulatory factors. Virulence factor regulator (Vfr) is a multifunctional DNA-binding regulator that modulates 2,4-DAPG biosynthesis in P. protegens FD6. However, the mechanism by which Vfr regulates this process remains unclear. In the present study, chromatin immunoprecipitation of FLAG-tagged Vfr and nucleotide sequencing analysis were used to identify 847 putative Vfr binding sites in P. protegens FD6. The consensus P. protegens Vfr binding site predicted from nucleotide sequence alignment is TCACA. The qPCR data showed that Vfr positively regulates the expression of phlF and phlG, and the expression of these genes was characterized in detail. The purified recombinant Vfr bound to an approximately 240-bp fragment within the phlF and phlG upstream regions that harbor putative Vfr consensus sequences. Using electrophoretic mobility shift assays, we localized Vfr binding to a 25-bp fragment that contains part of the Vfr binding region. Vfr binding was eliminated by mutating the TACG and CACA sequences in phlF and phlG, respectively. Taken together, our results show that Vfr directly regulates the expression of the 2,4-DAPG operon by binding to the upstream regions of both the phlF and phlG genes. However, unlike other Vfr-targeted genes, Vfr binding to P. protegens FD6 does not require an intact binding consensus motif. Furthermore, we demonstrated that vfr expression is autoregulated in this bacterium. These results provide novel insights into the regulatory role of Vfr in the biocontrol agent P. protegens.

Complete genome analysis of a newly isolated Shigella sonnei phage vB_SsoM_Z31

This work describes the characterization and genome annotation of a newly isolated lytic phage vB_SsoM_Z31 (referred to as Z31), isolated from wastewater samples collected in Dalian, China. Transmission electron microscope revealed that phage Z31 belongs to the family Myoviridae, order Caudovirales. This phage specifically infects the Shigella sonnei, Shigella dysenteriae and Escherichia coli. The genome of the phage Z31 is an 89,355 bp length dsDNA molecule with a G + C content of 38.87%. It has been predicted to contain 133 ORFs, and 24 tRNAs. No homologs of virulence factors or antimicrobial resistance genes were found in this phage. Based on the results of nucleotide sequence alignment and phylogenetic analysis, phage Z31 was assigned to the genus Felixounavirus, subfamily Ounavirnae.

Biological Characteristics and Molecular Mechanism of Procymidone-Resistance in Stemphylium eturmiunum from Garlic

Garlic leaf blight (GLB) caused by Stemphylium eturmiunum is first reported in Jiangsu Province of China. The dicarboximide fungicide (DCF) procymidone is reported to possess broad-spectrum action in inhibiting filamentous fungi and widely used to control leaf disease of various plants. In current study, of 41 S. eturmiunum isolates collected from commercial garlic farms in Pizhou and Dafeng Counties of Jiangsu Province, eight isolates were resistant to procymidone. Three phenotypes were categorized according to the in vitro responses to dicarboximide fungicides: S (sensitive), S+ (LR to iprodione and procymidone), and R2 (HR to all iprodione and procymidone). The fitness of all the resistant isolates was decreased in accordance with the data of mycelial growth, conidiation and virulence. After treated with 10µg/ml procymidone for 4 h, mycelial intracellular glycerol concentrations of resistant isolates were significantly lower than those of sensitive isolates. Positive cross-resistance was observed between dicarboximides and phenylpyrroles, but no cross-resistance between dicarboximides and fluazinam or difenoconazole in the two resistant phenotypes. Nucleotide sequence alignment results of the two-component histidine kinase genes from the sensitive and resistant isolates indicated that amino acid mutations were located at the histidine kinase, adenylyl cyclase, methyl-accepting chemotaxis protein, and phosphatase (HAMP) domain of N-terminal region, and response regulator domain (Rec) of C-terminal region. To our knowledge, the DCF-resistance in S. eturmiunum is first reported, and the findings are helpful to establish the rational strategy to manage the DCF-resistant populations of S. eturmiunum in field.

Characterisation and Mutagenesis Study of An Alternative Sigma Factor Gene (hrpL) from Erwinia mallotivora Reveal Its Central Role in Papaya Dieback Disease

The alternative sigma (σ) factor E, RpoE or HrpL, has been reported to be involved in stress- and pathogenicity-related transcription initiation in Escherichia coli and many other Gram-negative bacteria, including Erwinia spp. and Pseudomonas spp. A previous study identified the hrpL/rpoE transcript as one of the significant differentially expressed genes (DEGs) during early E. mallotivora infection in papaya and those data serve as the basis of the current project. Here, the full coding DNA sequence (CDS) of hrpL from E. mallotivora (EmhrpL) was determined to be 549 bp long, and it encoded a 21.3 kDa HrpL protein that possessed two highly conserved sigma-70 (σ70) motifs—σR2 and σR4. Nucleotide sequence alignment revealed the hrpL from E. mallotivora shared high sequence similarity to rpoE/hrpL from E. tracheiphila (83%), E. pyrifoliae (81%), and E. tasmaniensis (80%). Phylogenetics analysis indicated hrpL from E. mallotivora to be monophyletic with rpoEs/hrpLs from Pantoea vagans, E. herbicola, and E. tracheiphila. Structural analysis postulated that the E. mallotivora’s alternative σ factor was non-transmembranic and was an extracytoplasmic function (ECF) protein—characteristics shared by other σ factors in different bacterial species. Notably, the protein–protein interaction (PPI) study through molecular docking suggested the σ factor could be possibly inhibited by an anti-σ. Finally, a knockout of hrpL in E. mallotivora (ΔEmhrpL) resulted in avirulence in four-month-old papaya plants. These findings have revealed that the hrpL is a necessary element in E. mallotivora pathogenicity and also predicted that the gene can be inhibited by an anti-σ.

Genome analysis and phylogenetic characterization of two deformed wing virus strains from Apis cerana in Vietnam

Background Deformed wing virus (DWV) is a virulent virus that causes honeybee disease. DWV can exist as a latent infection in honeybees, outbreak into epidemics, and cause serious damage to beekeeping cross the world, including Vietnam. Methods The two DWV strains circulating in Vietnamese honeybee, Apis cerana, were first isolated from adult honeybees in North Vietnam (DWV-NVN) and South Vietnam (DWV-SVN). Their complete nucleotide sequences were determined, aligned, and compared with other DWV strains. Results The two Vietnamese DWV strains comprised 10,113 bp and contained a large single open reading frame (ORF) of 2,893 amino acids, initiating at nucleotide 1,130 and terminating at nucleotide 9,812. Multiple nucleotide sequence alignment between these two DWV-VN strains and DWV strains in A. mellifera was performed. The DWV-VN strains showed a low genetic identity (from 91.4% to 92.0%) with almost of these strains, but lower identities (89.2% and 89.4%) with UK2 and (89.6%) with the China2 strain. Low identities (91.7% and 91.9%) were also observed between the China3 strain (in A. cerana) and the DWV-VN strains, respectively. The deduced amino acid sequence alignment showed high genetic similarities (97.0%–97.9%) when the USA1, Chile, Italy1, France, UK1, UK2, Japan, Korea2, China1, China2 and China3 strains were compared to the DWV-VN strains. This ratio was 96.7% and 96.8% when the Korea1 strain was compared to the DWV-SVN and DWV-NVN strains, respectively. Numerous amino acid substitutions were identified in the L, VP3, and RdRp sequences. Notably, we observed six substitutions positioned at amino acids 27 (E > I), 98 (S > T), 120 (A > V), 153 (M > T), 170 (D > F), and 174 (Y > F) in the L protein, two amino acid changes at positions 980 (S > A) and 1032 (E > T) in VP3, and one amino acid change at position 2627 (R > C) unique to the DWV-VN strains. Phylogenetic analysis based on complete genome sequences, RdRp sequences and Simplot analysis indicated that there was a significant difference between DWV-VN strains in A. cerana and DWV strains in A. mellifera. The results suggested that the genetic variations of the DWV-VN strains in A. cerana help them to adapt geographical conditions and may lead to change the viral pathogenicity of DWV-VN strains.

DNA barcode of the Lanzones scale insect, Unaspis mabilis Lit & Barbecho (Hemiptera: Diaspididae)

The mitochondrial cytochrome c oxidase subunit 1 (COI) nucleotide sequences of Unaspis mabilis Lit & Barbecho (Hemiptera: Diaspididae), are provided for the first time. The total genomic DNA of each scale insect was extracted from individuals infesting lanzones leaves from three selected sites in Los Baños, Laguna. A partial COI gene amplicon with approximately 750 bp was obtained using the primer pair PcoF1 and LepR1. Nucleotide sequence alignment showed no variation among the COI sequences from all the samples. BLASTn search yielded no significant match with any of the available sequences for Unaspis species. The closest hit was Aulacaspis tubercularis Newstead (GenBank Accession No. HM474091) with 87.4% nucleotide similarity. Nonetheless, phylogenetic analyses revealed that generated COI sequences from U. mabilis form a monophyletic clade with U. yanonensis and U. euonymi, with closer proximity to the former. These findings also strengthen the species status of U. mabilis under the genus Unaspis. The DNA barcodes generated from this study (GenBank Acc. Nos. MN114099, MN14101, and MN114102), could, therefore, be used to verify the species identity of other lanzones scale accessions, as well as monitor the distribution and spread of U. mabilis, which would greatly influence possible pest management options. KEYWORDS: cytochrome C oxidase I, COI, Lansium domesticum Correa, lanzones

Isolation and Identification of Porcine Deltacoronavirus and Alteration of Immunoglobulin Transport Receptors in the Intestinal Mucosa of PDCoV-Infected Piglets

Porcine deltacoronavirus (PDCoV) is a porcine enteropathogenic coronavirus that causes watery diarrhea, vomiting, and frequently death in piglets, causing serious economic losses to the pig industry. The strain CHN-JS-2017 was isolated and identified by cytopathology, immunofluorescence assays, transmission electron microscopy, and sequence analysis. A nucleotide sequence alignment showed that the whole genome of CHN-JS-2017 is 97.4%–99.6% identical to other PDCoV strains. The pathogenicity of the CHN-JS-2017 strain was investigated in orally inoculated five-day-old piglets; the piglets developed acute, watery diarrhea, but all recovered and survived. CHN-JS-2017 infection-induced microscopic lesions were observed, and viral antigens were detected mainly by immunohistochemical staining in the small intestine. The neonatal Fc receptor (FcRn) and polymeric immunoglobulin receptor (pIgR) are crucial immunoglobulin (Ig) receptors for the transcytosis ofimmunoglobulin G (IgG), IgA, or IgM. Importantly, CHN-JS-2017 infected five-day-old piglets could significantly down-regulate the expression of FcRn, pIgR, and nuclear factor-kappa B (NF-κB)in the intestinal mucosa. Note that the level of FcRn mRNA in the intestinal mucosa of normal piglets is positively correlated with pIgR and NF-κB. At the same time, the expressions of FcRn, pIgR, and NF-κB mRNA are also positively correlated in infected piglets. These results may help explain the immunological and pathological changes associated with porcine deltacorononirus infection.

IN SILICO MODELING OF THE MOLECULAR STRUCTURE OF microRNAs MARKERS FOR LIVER FIBROSIS IN HEPATITIS C

Molecular biology looks for evidence that microRNA (miRNAs) plays a relevant function both in the beginning and advanced stages of hepatic fibrosis (HF), and has been proposed as an additional biomarker for HF forecasting in carriers of hepatitis C virus (HCV) infection. The purpose of this study was to develop an in silico modeling of the two-dimensional (2D) molecular structure of miRNA markers for HF in carriers of HCV. A search was initially performed for the nucleotide sequence of 6 miRNAs defined as biomarkers for HF, performinga computational simulation of the molecular structure of the following miRNAs: miRNA-182, miRNA-183, miRNA-1260b, miRNA-122-3p, miRNA-378i, and miRNA-214-5p. The nucleotide sequences were chosen in the GenBank of the American National Institutes of Health genetic sequence database. The nucleotide sequence alignment was carried out with a text-based format (FASTA) tool. In the molecular modeling, the structures were built with the RNAstructure, a completely automated miRNAs structure modelling server, available through Web Servers for RNA Secondary Structure Prediction. This study presented the nucleotide sequence and the computational simulation of molecular structures for the following miRNA: miRNA-182, miRNA-183, miRNA-1260b, miRNA-122-3p, miRNA-378i, and miRNA-214-5p. The molecular structure of miRNAs markers for HF in HCV carriers, through computational biology, is essential for designing more efficient optional tools for accurate treatment. KEY WORDS: micro-RNA; Hepatitis C; Hepatic Fibrosis; Computational Biology.

Characterisation of KiSS1R gene and Non genetic factors affecting reproductive traits in Indian goats

This study was undertaken to explore the genetic polymorphism in the KiSS1R (GPR54) gene from 80 Black Bengal, 50 Ganjam and 20 Raighar goat. Each of the sampled goats was recorded for its reproductive traits. The genomic DNA was isolated from the collected blood samples. The target 3’ UTR comprising of 246 bp fragment of KiSS1R gene was successfully amplified using the specific primer. Amplified samples were subjected for HRM analysis followed by sequencing. The nucleotide sequence alignment with the retrieved DNA sequence from NCBI BLAST confirmed absence of polymorphic pattern in KiSS1R gene in 3’ UTR. However, in the studied populations breed had significant effect on littersize, kidding interval and age at sexual maturity. It was found that age at sexual maturity and kidding interval were the highest in Ganjam goat population as compared to Raighar and Black Bengal goat population. Litter size was found highest in Black Bengal goat.