Genetic Variation Analysis on the Whole Genomic Sequence of a H9N2 Subtype Avian Influenza Virus Isolate

A Objective3 This study was to understand the genetic variation characters of the H9N2 subtype avian influenza virus isolate （A/Chicken/ Hebei/WD/98, abbreviated as WD98） by comparing with other reference strains. I-Method3 Eight complete genes were amplified by RT-PCR and sequenced. The homology and genetic evolution relationship were analyzed between these sequences and that of the seven reference strains. [Result] The whole genomic sequence of WD98 strain was 91.1% -95.8% homologous to that of seven reference strains tested. This isolate shared the highest homology （95.8%） to D/HK/Y280/97 and the lowest homology （91.1% ） to C/Pak/2/99. The HA cleavage site of the WD98 strain was R-S-S-R G, and the 226th amino acid at receptor-binding site was Gin. [ Condmion] WD98 strain belongs to mildly pathogenic avian in- fluenza virus and may not infect human. The genetic relationship is the closest between A/Chicken/Hebei/wD/98 and A/duck/HongKong/Y280/ 97, both of which belong to the sub-line of A/Chicken/Beijing/1/94 in Eurasian line. And A/Chicken/Hebei/WD/98 and A/Chicken/Beijing/1/94 are genetically distant within the same sub-line.

Genomic Sequence Determination of Classical Swine Fever Virus Persistent Infection Strain

Full genomic sequence of a newly isolated persistent infection strain of classical swine fever virus was firstly determined. It was demonstrated by sequence analyses that nucleotides homologies of this strain compared with virulent Shimen and vaccine HCLV were 89.7%and 87.7%, and homologies of amino acids were 94.8%and 93.3%, respectively. The sequencing results primarily suggest a tighter relationship between this persistent infection strain and virulent Shimen strain than vaccine HCLV strain.

The complete genomic sequence analysis of human norovirus NVgz01 strain in Guangzhou

The aim of this study is to explore the genomic molecular organization and genogroup of human nomvirus from infected infants in Guangzhou of China. Primers were designed according to the genomic sequence of norovims in the GenBank, and the nomvirus genome was amplified by RT-PCR. The PCR- products were cloned into T vector and sequenced, and the genomic nucleotide sequences were analyzed with the programs CLUSTAL W/X, DNASTAR and RAT （Recombination Analysis Tool）. The NVgz01 strain genome is 7558 bp in length and encodes three open reading frames （GenBank accession No. is DQ369797）. The genomic sequences of NVgz01 were compared with those of nomvirus in GenBank, which revealed that the homology with genogroup Ⅱ ranges between 76%-90%, and genogroup Ⅰ between 43%-44%. The ORF1 region shared 94% and 88% identity with Mc37 and Famiington strains, respectively; the capsid region （ORF2） shared 65% and 94% identity with Mc37 and Farmington strains, respectively. Phylogenetic trees were reconstructed by the neighbor-joining method. Comparative complete sequence analysis of the NVgz01 with reported human norovirus genomic sequences revealed that this isolate belongs to genogroup Ⅱ . The ORF1 and ORF2 regions shared different identity with Mc37 and Fannington strains, suggesting NVgz01 could be a recombinant virus.

The complete genomic sequence analysis of genotype 4 human astrovirus HASTVgz01 strain in Guangzhou

To analyze the genomic molecular structure and genotype of human astrovirus isolated from infant in Guangzhou of China, the primers were designed based on the genomic sequence of astrovirus from the C, enBank and the target sequence were amplified by RT-PCR. Then the PCR-products were cloned to T vector and sequenced. The genomic nucleotide sequences were analyzed by the programs CLUSTAL W and DNASTAR. It was found that the full genomic length of HASTVgz01 strain was 6721 bp and the ORFs were 6558 bp. The 5＇ and 3＇UTR were 82 and 81 nucleotides. The genome included 3 open reading frames （ORFs） ： ORFla, ORFlb and ORF2. The 5＇-terminal ORFla started at nueleotide 83 and extended to nucleotide 2845. ORFlb （nt 2785 to nt 4332） overlaped ORFla by 61 nueleotides. The 3＇-terminal ORF2 began at nucleotide 4325 and terminated at nucleotide 6640. ORF2 had 2316 nucleotides. Compared with other astrovirus sequences in GenBank, the homology of the amino acid sequence of ORF2 of HASTVgz01 strain with that of serotype 4 was 93%. Homology with other serotypes ranged from 61% to 70%. The complete nucleotide sequence of astrovirus HASTVgz01 strain isolated from Guangzhou in China was 6721 bp in length, GenBank accession NO. DQ344027. Comparing the ORF2 of astrovirus HASTVgz01 with the known sequences of types 1-8 the highest homology was serotype 4 （93%）. Comparative sequence analysis of the HASTVgz01 ORF2 with the reported human astrovirus sequences revealed that the isolated astrovirus belongs to genotype （serotype） 4.

The Application of Nicotiana benthamiana as a Transient Expression Host to Clone the Coding Sequences of Plant Genes

Coding sequences (CDS) are commonly used for transient gene expression, in yeast two-hybrid screening, to verify protein interactions and in prokaryotic gene expression studies. CDS are most commonly obtained using complementary DNA (cDNA) derived from messenger RNA (mRNA) extracted from plant tissues and generated by reverse transcription. However, some CDS are difficult to acquire through this process as they are expressed at extremely low levels or have specific spatial and/or temporal expression patterns in vivo. These challenges require the development of alternative CDS cloning technologies. In this study, we found that the genomic intron-containing gene coding sequences (gDNA) from Arabidopsis thaliana, Oryza sativa, Brassica napus, and Glycine max can be correctly transcribed and spliced into mRNA in Nicotiana benthamiana. In contrast, gDNAs from Triticum aestivum and Sorghum bicolor did not function correctly. In transient expression experiments, the target DNA sequence is driven by a constitutive promoter. Theoretically, a sufficient amount of mRNA can be extracted from the N. benthamiana leaves, making it conducive to the cloning of CDS target genes. Our data demonstrate that N. benthamiana can be used as an effective host for the cloning CDS of plant genes.

Improving the accuracy of genomic prediction for meat quality traits using whole genome sequence data in pigs

Background Pork quality can directly affect customer purchase tendency and meat quality traits have become valu-able in modern pork production.However,genetic improvement has been slow due to high phenotyping costs.In this study,whole genome sequence(WGS)data was used to evaluate the prediction accuracy of genomic best linear unbiased prediction(GBLUP)for meat quality in large-scale crossbred commercial pigs.Results We produced WGS data(18,695,907 SNPs and 2,106,902 INDELs exceed quality control)from 1,469 sequenced Duroc×(Landrace×Yorkshire)pigs and developed a reference panel for meat quality including meat color score,marbling score,L*(lightness),a*(redness),and b*(yellowness)of genomic prediction.The prediction accuracy was defined as the Pearson correlation coefficient between adjusted phenotypes and genomic estimated breeding values in the validation population.Using different marker density panels derived from WGS data,accuracy differed substantially among meat quality traits,varied from 0.08 to 0.47.Results showed that MultiBLUP outperform GBLUP and yielded accuracy increases ranging from 17.39%to 75%.We optimized the marker density and found medium-and high-density marker panels are beneficial for the estimation of heritability for meat quality.Moreover,we conducted genotype imputation from 50K chip to WGS level in the same population and found average concord-ance rate to exceed 95%and r^(2)=0.81.Conclusions Overall,estimation of heritability for meat quality traits can benefit from the use of WGS data.This study showed the superiority of using WGS data to genetically improve pork quality in genomic prediction.

The evolution of cancer genomic medicine in Japan and the role of the National Cancer Center Japan

The journey to implement cancer genomic medicine(CGM)in oncology practice began in the 1980s,which is considered the dawn of genetic and genomic cancer research.At the time,a variety of activating oncogenic alterations and their functional significance were unveiled in cancer cells,which led to the development of molecular targeted therapies in the 2000s and beyond.Although CGM is still a relatively new discipline and it is difficult to predict to what extent CGM will benefit the diverse pool of cancer patients,the National Cancer Center(NCC)of Japan has already contributed considerably to CGM advancement for the conquest of cancer.Looking back at these past achievements of the NCC,we predict that the future of CGM will involve the following:1)A biobank of paired cancerous and non-cancerous tissues and cells from various cancer types and stages will be developed.The quantity and quality of these samples will be compatible with omics analyses.All biobank samples will be linked to longitudinal clinical information.2)New technologies,such as whole-genome sequencing and artificial intelligence,will be introduced and new bioresources for functional and pharmacologic analyses(e.g.,a patient-derived xenograft library)will be systematically deployed.3)Fast and bidirectional translational research(bench-to-bedside and bedside-to-bench)performed by basic researchers and clinical investigators,preferably working alongside each other at the same institution,will be implemented;4)Close collaborations between academia,industry,regulatory bodies,and funding agencies will be established.5)There will be an investment in the other branch of CGM,personalized preventive medicine,based on the individual's genetic predisposition to cancer.

Computational analysis and prediction for exons of PAC579 genomic sequence

To isolate the novel genes related to human hepatocellular carcinoma (HCC), we sequenced P1-derived artificial chromosome PAC579 (D17S926 locus) mapped in the minimum LOH (loss of heterozygosity) deletion region of chromosome 17p13.3 in HCC, Four novel genes mapped in this genomic sequence area were isolated and cloned by wet-lab experiments, and the exons of these genes were located. 0-60 kb of this genomic sequence including the genes of interest was scanned with five different computational exon prediction programs as well as four splice site recognition programs. After analyzing and comparing the computationally predicted results with the wet-lab experiment results, some potential exons were predicted in the genomic sequence by using these programs.

Breed identification using breed‑informative SNPs and machine learning based on whole genome sequence data and SNP chip data

Background Breed identification is useful in a variety of biological contexts.Breed identification usually involves two stages,i.e.,detection of breed-informative SNPs and breed assignment.For both stages,there are several methods proposed.However,what is the optimal combination of these methods remain unclear.In this study,using the whole genome sequence data available for 13 cattle breeds from Run 8 of the 1,000 Bull Genomes Project,we compared the combinations of three methods(Delta,FST,and In)for breed-informative SNP detection and five machine learning methods(KNN,SVM,RF,NB,and ANN)for breed assignment with respect to different reference population sizes and difference numbers of most breed-informative SNPs.In addition,we evaluated the accuracy of breed identification using SNP chip data of different densities.Results We found that all combinations performed quite well with identification accuracies over 95%in all scenarios.However,there was no combination which performed the best and robust across all scenarios.We proposed to inte-grate the three breed-informative detection methods,named DFI,and integrate the three machine learning methods,KNN,SVM,and RF,named KSR.We found that the combination of these two integrated methods outperformed the other combinations with accuracies over 99%in most cases and was very robust in all scenarios.The accuracies from using SNP chip data were only slightly lower than that from using sequence data in most cases.Conclusions The current study showed that the combination of DFI and KSR was the optimal strategy.Using sequence data resulted in higher accuracies than using chip data in most cases.However,the differences were gener-ally small.In view of the cost of genotyping,using chip data is also a good option for breed identification.

Complete Sequence and Gene Organization of the Mitochondrial Genome of Tokay (Gekko gecko)

Long-PCR amplification, clone and primer-walking sequencing methods were employed in determine the complete sequence of mitochondrial genome of tokay (Gekko gecko). The genome is 16 435 bp in size, contains 13 protein-coding, 2 ribosomal and 22 transfer RNA genes. The mt genome of Gekko is similar to most of the vertebrates in gene components, order, orientation, tRNA structures, low percentage of guanine and high percentage of thymine, and skews of base GC and AT. Base A was preferred at third codon positions for protein genes is similar to amphibians and fishes rather than amnion vertebrates. The standard stop codes (TAA) present only in three protein genes, less than those of most vertebrates. Transfer RNA genes range in length from 63 to 76 nt, their planar structure present characteristic clover leaf, except for tRNA-Cys and tRNA-Ser (AGY) because of lacking the D arm.

Complete Nucleotide Sequence of a Newly Avirulent Newcastle Disease Virus Hubei 92(HB92) Strain

A new avirulent, heat-resistance HB92 strain of newcastle Disease Virus (NDV) was acquired from Australia V4 strain. Its complete nucleotides sequence was first determined. The entire genome of NDV HB92 consists of 15 186 nucleotides (GenBank accession no. AY225110). It was demonstrated by sequence analysis that nucleotides homology of HB92 strain with virulent strain ZJ1 were 83.9%, and the homology compared with avirulent vaccine strain La Sota and B1 were 94.0% and 93.5%, respectively. These results might be contributive to the study of the molecular mechanism of evolution of the NDV strain HB92 and to develop the engineered recombinant vaccine. Key words newcastle disease virus - genomic sequence - sequence analysis CLC number S 852. 65 Foundation item: Supported by Hubei Natural Science Foundation (2002AB144)Biography: Pan Zhi-shu(1961-), male, Ph. D, Associate professor, research direction: molecular biology and pathogenesis of eucaryotic viruses.

Identification of novel genes associated with atherosclerosis in Bama miniature pig

Background:Atherosclerosis is a chronic cardiovascular disease of great concern.However,it is difficult to establish a direct connection between conventional small animal models and clinical practice.The pig's genome,physiology,and anatomy reflect human biology better than other laboratory animals,which is crucial for studying the pathogenesis of atherosclerosis.Methods:We used whole-genome sequencing data from nine Bama minipigs to perform a genome-wide linkage analysis,and further used bioinformatic tools to filter and identify underlying candidate genes.Candidate gene function prediction was performed using the online prediction tool STRING 12.0.Immunohistochemistry and immunofluorescence were used to detect the expression of proteins encoded by candidate genes.Results:We mapped differential single nucleotide polymorphisms(SNPs)to genes and obtained a total of 102 differential genes,then we used GO and KEGG pathway enrichment analysis to identify four candidate genes,including SLA-1,SLA-2,SLA-3,and TAP2.nsSNPs cause changes in the primary and tertiary structures of SLA-I and TAP2 proteins,the primary structures of these two proteins have undergone amino acid changes,and the tertiary structures also show slight changes.In addition,immunohistochemistry and immunofluorescence results showed that the expression changes of TAP2 protein in coronary arteries showed a trend of increasing from the middle layer to the inner layer.Conclusions:We have identified SLA-I and TAP2 as potential susceptibility genes of atherosclerosis,highlighting the importance of antigen processing and immune response in atherogenesis.

Isolation and Characterization of SARS-CoV-2 in Kenya

The discovery of Severe Acute Respiratory Syndrome-Coronavirus-2 (SARS-CoV-2) in Wuhan, Hubei province, China, in December 2019 raised global health warnings. Quickly, in 2020, the virus crossed borders and infected individuals across the world, evolving into the COVID-19 pandemic. Notably, early signs of the virus’s existence were observed in various countries before the initial outbreak in Wuhan. As of 12th of April, the respiratory disease had infected over 762 million people worldwide, with over 6.8 million deaths recorded. This has led scientists to focus their efforts on understanding the virus to develop effective means to diagnose, treat, prevent, and control this pandemic. One of the areas of focus is the isolation of this virus, which plays a crucial role in understanding the viral dynamics in the laboratory. In this study, we report the isolation and detection of locally circulating SARS-CoV-2 in Kenya. The isolates were cultured on Vero Cercopithecus cell line (CCL-81) cells, RNA extraction was conducted from the supernatants, and reverse transcriptase-polymerase chain reaction (RT-PCR). Genome sequencing was done to profile the strains phylogenetically and identify novel and previously reported mutations. Vero CCL-81 cells were able to support the growth of SARS-CoV-2 in vitro, and mutations were detected from the two isolates sequenced (001 and 002). Genome sequencing revealed the circulation of two isolates that share a close relationship with the Benin isolate with the D614G common mutation identified along the S protein. These virus isolates will be expanded and made available to the Kenya Ministry of Health and other research institutions to advance SARS-CoV-2 research in Kenya and the region.

To Analyze the Sensitivity of RT-PCR Assays Employing S Gene Target Failure with Whole Genome Sequencing Data during Third Wave by SARS-CoV-2 Omicron Variant

Introduction: Omicron is a highly divergent variant of concern (VOCs) of a severe acute respiratory syndrome SARS-CoV-2. It carries a high number of mutations in its spike protein hence;it is more transmissible in the community by immune evasion mechanisms. Due to mutation within S gene, most Omicron variants have reported S gene target failure (SGTF) with some commercially available PCR kits. Such diagnostic features can be used as markers to screen Omicron. However, Whole Genome Sequencing (WGS) is the only gold standard approach to confirm novel microorganisms at genetically level as similar mutations can also be found in other variants that are circulating at low frequencies worldwide. This Retrospective study is aimed to assess RT-PCR sensitivity in the detection of S gene target failure in comparison with whole genome sequencing to detect variants of Omicron. Methods: We have analysed retrospective data of SARS-CoV-2 positive RT-PCR samples for S gene target failure (SGTF) with TaqPath COVID-19 RT-PCR Combo Kit (ThermoFisher) and combined with sequencing technologies to study the emerged pattern of SARS-CoV-2 variants during third wave at the tertiary care centre, Surat. Results: From the first day of December 2021 till the end of February 2022, a total of 321,803 diagnostic RT-PCR tests for SARS-CoV-2 were performed, of which 20,566 positive cases were reported at our tertiary care centre with an average cumulative positivity of 6.39% over a period of three months. In the month of December 21 samples characterized by the SGTF (70/129) were suggestive of being infected by the Omicron variant and identified as Omicron (B.1.1.529 lineage) when sequence. In the month of January, we analysed a subset of samples (n = 618) with SGTF (24%) and without SGTF (76%) with Ct values Conclusions: During the COVID-19 pandemic, it took almost more than 15 days to diagnose infection and identify pathogen by sequencing technology. In contrast to that molecular assay provided quick identification with the help of SGTF phenomenon within 5 hours of duration. This strategy helps scientists and health policymakers for the quick isolation and identification of clusters. That ultimately results in a decreased transmission of pathogen among the community.

Epidemiology and Clonal Spread Evidence of Carbapenem-Resistant Organisms in the Center of Care and Protection of Orphaned Children, Vietnam

Objective: To determine the prevalence of colonization and transmission of carbapenem-resistant Gram-negative organisms in order to develop of an effective infection prevention program. Design: Cross-sectional study with carbapenem-resistant organisms (CRO) colonization detection from the fecal specimens of 20 Health Care Workers (HCWs) and 67 residents and 175 random environment specimens from September 2022 to September 2023. Setting: A Care and Protection Centre of Orphaned Children in South of HCM City. Participants: It included 20 HCWs, 67 residents, and 175 randomly collected environmental specimens. Method: Rectal and environmental swabs were collected from 20 HCWs, 67 residents (most of them were children), and 175 environmental specimens. MELAB Chromogenic CARBA agar plates, Card NID, and NMIC-500 CPO of the BD Phoenix TM Automated Microbiology System and whole genome sequencing (WGS) were the tests to screen, confirm CROs, respectively and determine CRO colonization and transmission between HCWs, residents, and the environment. Result: We detected 36 CRO isolates, including 6, 11 and 19 CROs found in 6 HCWs, 10 residents and 19 environments. The prevalence of detectable CRO was 30% (6/20) in HCWs, 14.92% (10/67) in residents, and 10.86% (19/175) in environmental swabs in our study. WGS demonstrated CRO colonization and transmission with the clonal spread of E. coli and A. nosocomialis, among HCWs and residents (children). Conclusion: Significant CRO colonization and transmission was evident in HCWs, residents, and the center environment. Cleaning and disinfection of the environment and performing regular hand hygiene are priorities to reduce the risk of CRO colonization and transmission.

Complete genome sequences of four isolates of Citrus leaf blotch virus from citrus in China

Citrus leaf blotch virus （CLBV） is a member of the genus Citrivirus, in the family Betaflexiviridae. It has been reported CLBV could infect kiwi, citrus and sweet cherry in China. Of 289 citrus samples from six regions of China, 15 were detected to be infected with CLBV in this study. The complete genome of four isolates of CLBV was obtained from Reikou in Sichuan （CLBV-LH）, Yura Wase in Zhejiang （CLBV-YL）, Bingtangcheng in Hunan （CLBV-BT）, Fengjie 72-1 in Chongqing （CLBV- F J）, respectively. While they all represented 8 747 nucleotides in monopartite size, excluding the poly（A） tail, each of the isolates coded three open reading frames （ORFs）. Identity of the four isolates ranged from 98.9 to 99.8% to each other and from 96.8 to 98.1% to the citrus references in GenBank by multiple alignment of genomes. A phylogenetic tree based on the genome sequences of available CLBV isolates indicated that the four isolates were clustered together, suggesting that CLBV isolates from citrus in China did not have obvious variation. This is the first report of the complete nucleotide sequences of CLBV isolates infecting citrus in China.

Complete Genome Sequence of Mycoplasma ovipneumoniae Strain NM2010, Which Was Isolated from a Sheep in China

Mycoplasma ovipneumoniae, a kind of mycoplasma bacteria, commonly infects the respiratory tract causing respiratory disease in sheep and goats worldwide. Here, the complete genome sequence of M. ovipneumoniae strain NM2010 isolated from a sheep in China was reported for the ifrst time.

The complete genome sequence of Citrus vein enation virus from China

The complete nucleotide sequence of an isolate of Citrus vein enation virus（CVEV-XZG） from China has been determined for the first time. The genome consisted of 5 983 nucleotides, coding for five open reading frames（ORFs）, had a similar genomic organization features with Pea enation mosaic virus（PEMV）. Nucleotide and deduced amino acid sequence identity of the five ORFs compared to isolate CVEV VE-1 range from 97.1 to 99.0% and 97.4 to 100.0%, these values compared to isolate PEMV-1 range from 45.2 to 51.6% and 31.1 to 45.2%. Phylogenetic analysis based on the complete genome sequence showed that the isolate CVEV-XZG had close relationship with Pea enation mosaic virus. The results supports CVEV may be a new member of genus Enamovirus. The full sequence of CVEV-XZG presented here may serve as a basis for future study of CVEV in China.

Characterization and Genomic Analysis of a Plaque Purified Strain of Cyanophage PP

Cyanophages are ubiquitous and essential components of the aquatic environment and play an important role in the termination of algal blooms.As such,they have attracted widespread interest.PP was the first isolated cyanophage in China,which infects Plectonema boryanum and Phormidium foveolarum.In this study,this cyanophage was purified three times by a double-agar overlay plaque assay and characterized.Its genome was extracted,totally sequenced and analyzed.Electron microscopy revealed a particle with an icosahedral head connected to a short stubby tail.Bioassays showed that PP was quite virulent.The genome of PP is a 42,480 base pair(bp),linear,double-stranded DNA molecule with 222 bp terminal repeats.It has high similarity with the known Pf-WMP3 sequence.It contains 41 open reading frames(ORFs),17 of which were annotated.Intriguingly,the genome can be divided into two completely different parts,which differ both in orientation and function.

Complete genome sequence and proteomic analysis of a thermophilic bacteriophage BV1

Viruses of thermophiles are of great interest due to their roles in gene transfer, global geochemical cycle and evolution of life on earth. However, the thermophilic bacteriophages have not been studied extensively. In this investigation, a typical bacteriophage BV1 was obtained from a thermophilic bacterium Geobacillus sp. 6k512, which was isolated from an inshore hot spring in Xiamen of China. The BV1 contained a double-stranded linear DNA of 35 055 bp, which encodes 54 open reading frames （ORFs）. Interestingly, eight of the 54 BV1 ORFs shared sequence similarities to genes from human disease-relevant bacteria. Seven proteins of the purified BV1 virions were identified by proteomic analysis. Determination of BV1 functional genomics would facilitate the better understanding of the mechanism for virus-thermophile interaction.