Structure-function relationships among selected human coronaviruses-Supplementary data

Identifying the key proteins among different types of human disease-causing coronaviruses is essential for the molecular mechanism and thereby designing potential drug molecules. Eight selected proteins of seven types of disease-causing coronaviruses, viz.SARS-CoV-2 (severe acute respiratory syndrome coronavirus2), SARS-CoV (severe acute respiratory syndrome coronavirus), MERS-CoV (middle east respiratory syndrome coronavirus), Human coronavirus OC43, Human coronavirus HKU1, Human coronavirus 229E and Human coronavirus NL63, were chosen for the comparison. Further, an attempt has been made to explore the most important host-pathogen interactions with a special focus on spike (RBD) protein region as this region deemed to be functionally most important. Epitope region was also identified which helps in the design of epitope-based vaccines. The structural comparison carried out among the seven types of human coronaviruses has revealed the molecular level details on the similarity among this series. This study has facilitated the identification of the important residues in the studied proteins which control the key functions such as viral replication and transmission. Thus, exploring the protein space in the family of coronaviruses, provide valuable insights into the molecular basis associated with the role of proteins and viral infections, which is expected to trigger the identification of the drug targets for coronaviruses infections, in a rational way.

Structure-function relationships among selected human coronaviruses-Supplementary data

Identifying the key proteins among different types of human disease-causing coronaviruses is essential for the molecular mechanism and thereby designing potential drug molecules. Eight selected proteins of seven types of disease-causing coronaviruses, viz.SARS-CoV-2 (severe acute respiratory syndrome coronavirus2), SARS-CoV (severe acute respiratory syndrome coronavirus), MERS-CoV (middle east respiratory syndrome coronavirus), Human coronavirus OC43, Human coronavirus HKU1, Human coronavirus 229E and Human coronavirus NL63, were chosen for the comparison. Further, an attempt has been made to explore the most important host-pathogen interactions with a special focus on spike (RBD) protein region as this region deemed to be functionally most important. Epitope region was also identified which helps in the design of epitope-based vaccines. The structural comparison carried out among the seven types of human coronaviruses has revealed the molecular level details on the similarity among this series. This study has facilitated the identification of the important residues in the studied proteins which control the key functions such as viral replication and transmission. Thus, exploring the protein space in the family of coronaviruses, provide valuable insights into the molecular basis associated with the role of proteins and viral infections, which is expected to trigger the identification of the drug targets for coronaviruses infections, in a rational way.

Structure-function relationships among selected human coronaviruses

Identifying the key proteins among different types of human disease-causing coronaviruses is essential for the molecular mechanism and thereby designing potential drug molecules. Eight selected proteins of seven types of disease-causing coronaviruses, viz.SARS-CoV-2 (severe acute respiratory syndrome coronavirus2), SARS-CoV (severe acute respiratory syndrome coronavirus), MERS-CoV (middle east respiratory syndrome coronavirus), Human coronavirus OC43, Human coronavirus HKU1, Human coronavirus 229E and Human coronavirus NL63, were chosen for the comparison. Further, an attempt has been made to explore the most important host-pathogen interactions with a special focus on spike (RBD) protein region as this region deemed to be functionally most important. Epitope region was also identified which helps in the design of epitope-based vaccines. The structural comparison carried out among the seven types of human coronaviruses has revealed the molecular level details on the similarity among this series. This study has facilitated the identification of the important residues in the studied proteins which control the key functions such as viral replication and transmission. Thus, exploring the protein space in the family of coronaviruses, provide valuable insights into the molecular basis associated with the role of proteins and viral infections, which is expected to trigger the identification of the drug targets for coronaviruses infections, in a rational way.

APLICACIÓN DEL MODELO DE PÉRDIDA DE ADN PARA EL DISEÑO DE CEBADORES EN Potamotrygon magdalenae (POTAMOTRYGONIDAE) / Application of the model of DNA loss for desing of primers in Potamotrygon magdalenae (Potamotrygonidae)

RESUMEN El diseño de cebadores es fundamental para amplificar regiones de genes debido a que la especificidad que mantienen cebador-secuencia de interés puede causar el éxito o fracaso en la reacción de PCR. En relación a Potamotrygon magdalenae (especie de interés de acuerdo al PAN Tiburones-Colombia), existe poca información disponible de aspectos relacionados con la genética poblacional de esta Raya. El objetivo del presente trabajo consistió en diseñar cebadores bajo los criterios del Modelo de Pérdida de ADN (DNA-LM), que permitan evaluar el estado genético de las poblaciones de P. magdalenae. Alineamos secuencias de la superfamilia Dayastoidea, disponibles en el NCBI, de los genes mitocondriales Citocromo C Oxidasa 1 (MT-CO1) y Citocromo b (MT-CYB). Seguimos los parámetros Gap open penalty (5), Gap extension penalti (0,2) y Terminalgap penalties (0,1) y seleccionamos dos pares de cebadores de acuerdo con el DNA-LM. Estimamos el producto amplificado del gen MT-CO1 en 916 pb y del gen MT-CYB en 774 pb, en muestras de P. magdalenae procedentes de diferentes ciénagas del Magdalena medio. Discutimos los resultados desde la perspectiva de validar la especificidad de los cebadores diseñados, teniendo en cuenta la correspondencia e identidad de las secuencias de los genes considerados. Los cebadores aquí reportados pueden contribuir a ampliar el conocimiento de la genética poblacional, biogeografía y filogenética de la raya de agua dulce P. magdalenae.

ABSTRACT The primer design is critical for amplifying gene regions because the specificity that primer-sequence maintain can cause success or failure in the PCR reaction. Concerning Potamotrygon magdalenae (a species of interest according to the PAN Tiburones-Colombia), there is little information available about aspects related to population genetics of this river stingray. This study aimed to design primers under DNA Loss Model (DNA-LM) criteria, which will allow assessing the genetics status of populations of P. magdalenae. We aligned sequences from the superfamily Dayastoidea, available in the NCBI, of the mitochondrial genes Cytochrome C Oxidase 1 (MT-CO1) and Cytochromeb (MT-CYB). We followed the parameters Gap open penalty (5), Gap extension penalty (0.2) and Terminal gap penalties (0.1) and selected two pairs of primers according to the DNA-LM. We calculated the amplified product of the MT-CO1 gene in 916 pb and the MT-CYB gene in 774 pb in samples from different swamps of the middle Magdalena. We discussed the results from the perspective of validating the specificity of the primers designed, considering the correspondence and identity of gene sequences considered. The primers reported here will contribute to broadening the knowledge of population genetics, biogeography and phylogenetic of the river stingray P. magdalenae.

From the Origins of SARS-CoV-2 to the Correct Application of BLAST / 中国生物化学与分子生物学报

Basic local alignment search tool (BLAST) is one of the popular sequence similarity analysis tools. However, some students and researchers just blindly use the default parameters. Moreover, some students are confused about how to choose the right program. In a word, it is prone to be misused and researchers often draw conclusions incorrectly. In view of this, we traced back the internet hot topic in early 2020 - "MORDERATELY STRONG CONFIRMATION OF A LABORATORY ORIGIN OF COVID-19", and took it as teaching materials to guide the student to use BLAST currently through reanalyzing and reproducing the source of errors. Then we arranged an interesting experiment about fabricating dinosaur genes through modifying a chicken gene. In the experimental design to make the students grasp the BLAST tools better, one group fabricated the dinosaur gene and the other group decrypted the added bases. This instructional design could be conducive to cultivate students ' ability about distinguishing different viewpoints correctly, and we hope it can be enlightening and helpful to the teaching of BLAST tools.

Genomic characteristics of 2019 novel coronavirus from imported cases of coronavirus disease 2019 in Henan Province / 中华传染病杂志

Objective:To analyze the evolutionary characteristics and variations of 2019 novel coronavirus (2019-nCoV) strains imported from abroad in Henan Province.Methods:A total of 16 imported cases of coronavirus disease 2019 (COVID-19) reported in Henan Province from May to December 2020 were enrolled. The throat swab specimens from the patients were collected and sent to the Henan Provincial Center for Disease Control and Prevention for whole genome sequencing. Taking SARS-CoV-2 Wuhan-Hu-1 published in Global Initiative on Sharing All Influenza Data (GISAID) as the reference sequence, the sequences were aligned and analyzed by MEGA X, and the phylogenetic tree was constructed by the maximum likelihood method.Results:Among 16 cases, 13 cases were imported from Russia, two cases were imported from Myanmar, and one case was imported from Ukraine. A total of 16 strains of 2019-nCoV genomes with the lengths of 29 804 bp to 29 882 bp were obtained. A total of 145 nucleotide mutations and 80 amino acid mutations were detected. Nucleotide variations of C241T, C3037T, C14408T, A23403G and the amino acid variation of D614G in spike protein were detected in all sequences. Meanwhile, insertion A at the site of 29704 was found in BetaCov/HEN02/Human/2020, BetaCov/HEN04/Human/2020 and BetaCov/HEN05/Human/2020. Deletion variation was not found. Phylogenetic analysis showed that there was no correlation between the 16 strains and currently epidemic variants of concern (VOC) .Conclusion:From May to December 2020, the detection of viral genome mutations in the imported cases of Henan Province shows randomness and diversity, while the strains are not VOC.

Homologous modeling and binding ability analysis of Spike protein after point mutation of severe acute respiratory syndrome coronavirus 2 to receptor proteins and potential antiviral drugs / 北京大学学报(医学版)

OBJECTIVE@#To explore the natural mutations in Spike protein (S protein) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the changes of affinity between virus and associated receptors or drug molecules before and after the mutation based on whole length sequencing results.@*METHODS@#In the study, the bioinformatics analysis of all the published sequences of SARS-CoV-2 was conducted and thus the high frequency mutation sites were affirmed. Taking advantages of PolyPhen-2, the functional influence of each mutation in S protein was prospected. The 3D homologous modelling was performed by SWISS-MODEL to establish mutated S protein structural model, in which the protein-docking was then implemented with angiotensin-converting enzyme 2 (ACE2), dipeptidyl peptidase-4 (DPP4) and aminopeptidase N (APN) by ZDOCK, and the combining capacity of each mutated S protein evaluated by FiPD. Finally, the binding ability between mutated S proteins and anti-virus drugs were prospected and evaluated through AutoDock-Chimera 1.14.@*RESULTS@#The mutations in specific region of S protein had greater tendency to destroy the S protein function by analysis of mutated S protein structure. Protein-receptor docking analysis between naturally mutated S protein and host receptors showed that, in the case of spontaneous mutation, the binding ability of S protein to ACE2 tended to be weakened, while the binding ability of DPP4 tended to be enhanced, and there was no significant change in the binding ability of APN. According to the computational simulation results of affinity binding between small molecular drugs and S protein, the affinity of aplaviroc with S protein was significantly higher than that of other small molecule drug candidates.@*CONCLUSION@#The region from 400-1 100 amino acid in S protein of SARS-CoV-2 is the mutation sensitive part during natural state, which was more potential to mutate than other part in S protein during natural state. The mutated SARS-CoV-2 might tend to target human cells with DPP4 as a new receptor rather than keep ACE2 as its unique receptor for human infection. At the same time, aplaviroc, which was used for the treatment of human immunodeficiency virus (HIV) infection, may become a new promising treatment for SARS-CoV-2 and could be a potential choice for the development of SARS-CoV-2 drugs.

Identification of differentially expressed genes in lesional versus nonlesional skin of patients with atopic dermatitis by using high-throughput transcriptome-wide RNA sequencing / 中华皮肤科杂志

Objective@#To identify differentially expressed genes in the transcriptome of the lesional versus nonlesional skin tissues of patients with moderate and severe atopic dermatitis (AD) , and to elucidate their roles in the pathogenesis of AD.@*Methods@#From July to October in 2016, lesional and nonlesional skin tissues were obtained from 5 outpatients of Han nationality with AD in Guangzhou Institute of Dermatology, Institute of Dermatology, Guangzhou Medical University. The next-generation high-throughput transcriptome-wide RNA sequencing (RNA-seq) was performed to identify differentially expressed genes, which were subjected to GO function annotation and KEGG pathway analysis. Real-time fluorescence-based quantitative PCR (qRT-PCR) was conducted to verify differences in candidate gene expression between lesional and nonlesional skin tissues.@*Results@#An average of 10.96 GBs sequence reads were acquired among 10 samples. A total of 21 729 genes were detected, including 19 268 known genes and 2 545 predicted novel genes. A total of 23 153 new transcripts were detected, of which 18 889 were new alternative splicing subtypes of known protein-coding genes, 2 545 were transcripts belonging to new protein-coding genes, and the remaining 1 719 belonged to long-stranded non-coding RNA. Totally, 78 differentially expressed genes were identified between the lesional and nonlesional skin tissues, including 69 upregulated and 11 downregulated genes in the lesional skin tissues. Among them, there were several genes known to be associated with AD inflammation (CXCL1/2/8, IL6/IL1β, MMP1, SERPINB4, S100A2, GZMB, OASL, OSM) and barrier (KRT16, FABP5, CYP1A1) and keratinocyte differentiation (IL-20) . GO analysis revealed that functions of 72 differentially expressed genes could be annotated. KEGG pathway analysis showed that the differentially expressed genes were grouped into 132 signaling pathways, of which 13 were significantly enriched, including the interleukin (IL) -17 pathway, NOD-like receptor signaling pathway, Toll-like receptor signaling pathway, etc. qRT-PCR showed that the mRNA expression levels of candidate genes CXCL1, KRT6A, IL36A, SERPINB4 and PSAPL1 was consistent with the transcriptome sequencing results.@*Conclusions@#Differentially expressed genes and related important regulatory signaling pathways were identified between the lesional and nonlesional skin tissues of patients with AD at the transcriptional level, and the IL-17 pathway was found to be mostly enriched in AD lesions in patients of Han nationality. These findings provide an important basis for further study on the pathogenesis of AD..

Identification of differentially expressed genes in lesional versus nonlesional skin of patients with atopic dermatitis by using high-throughput transcriptome-wide RNA sequencing / 中华皮肤科杂志

Objective To identify differentially expressed genes in the transcriptome of the lesional versus nonlesional skin tissues of patients with moderate and severe atopic dermatitis(AD), and to elucidate their roles in the pathogenesis of AD. Methods From July to October in 2016, lesional and nonlesional skin tissues were obtained from 5 outpatients of Han nationality with AD in Guangzhou Institute of Dermatology, Institute of Dermatology, Guangzhou Medical University. The next-generation high-throughput transcriptome-wide RNA sequencing (RNA-seq) was performed to identify differentially expressed genes, which were subjected to GO function annotation and KEGG pathway analysis. Real-time fluorescence-based quantitative PCR(qRT-PCR)was conducted to verify differences in candidate gene expression between lesional and nonlesional skin tissues. Results An average of 10.96 GBs sequence reads were acquired among 10 samples. A total of 21729 genes were detected, including 19268 known genes and 2545 predicted novel genes. A total of 23153 new transcripts were detected, of which 18889 were new alternative splicing subtypes of known protein-coding genes, 2545 were transcripts belonging to new protein-coding genes, and the remaining 1719 belonged to long-stranded non-coding RNA. Totally, 78 differentially expressed genes were identified between the lesional and nonlesional skin tissues, including 69 upregulated and 11 downregulated genes in the lesional skin tissues. Among them, there were several genes known to be associated with AD inflammation (CXCL1/2/8, IL6/IL1β, MMP1, SERPINB4, S100A2, GZMB, OASL, OSM) and barrier (KRT16, FABP5, CYP1A1) and keratinocyte differentiation (IL-20). GO analysis revealed that functions of 72 differentially expressed genes could be annotated. KEGG pathway analysis showed that the differentially expressed genes were grouped into 132 signaling pathways, of which 13 were significantly enriched, including the interleukin(IL)-17 pathway, NOD-like receptor signaling pathway, Toll-like receptor signaling pathway, etc. qRT-PCR showed that the mRNA expression levels of candidate genes CXCL1, KRT6A, IL36A, SERPINB4 and PSAPL1 was consistent with the transcriptome sequencing results. Conclusions Differentially expressed genes and related important regulatory signaling pathways were identified between the lesional and nonlesional skin tissues of patients with AD at the transcriptional level, and the IL-17 pathway was found to be mostly enriched in AD lesions in patients of Han nationality. These findings provide an important basis for further study on the pathogenesis of AD. .

Identification of pathogenic nonsporulating molds in eyes by internal transcribed spacer sequences alignment / 国际眼科杂志(Guoji Yanke Zazhi)

@#AIM: To identify the species and genus of pathogenic nonsporulating molds(NSM)by internal transcribed spacer(ITS)sequences alignment, and reveal the biodiversity of NSM in Hainan Island with tropical climate. <p>METHODS: Nine teen fungal strains, identified as NSM by conventional method in the laboratory department of Hainan Province Eye Hospital, were involved in this study. All of the strains were isolated from the infectious eye tissues from patients, and cultured with potato-dextrose agar and Sabourand's agar in 37℃ incubator for 7-21d. Any reproductive structure was detected by microscopy for up to 21d. After growth for 3-7d, the genomic DNA of specimens were extracted by grinding method combined with chemical method. Then, ITS sequences in the ribosome were amplified by PCR and analyzed using the National Center for Biological Information(NCBI)GenBank database. Finally, the species and genus were confirmed by sequence alignment. <p>RESULTS: The corresponding target bars could be observed in all 19 specimens after PCR. The results from genetic alignment classified the 19 specimens into 12 species, including Lasiodiplodia theobromae(6 isolates), Curvalaria lunata(1 isolate), Arthrinium sp.(2 isolates), neodeightonia subglabosa(2 isolates), Earliella scabrosa(1 isolate), Hypocreales sp.(1 isolate), phoma multirostrata(1 isolate), Trichophyton rubrum(1 isolate), Aspergillus westerdijkiae(1 isolate), roussoella siamensis(1 isolate), Ceriporia lacerata(1 isolate), Fusarium solqni(1 isolate).<p>CONCLUSION: ITS sequence alignment can identify NSM to genus and species level. The NSM in Hainan Island contains varies species, and is associated with multiple infectious diseases of the eye.

Study on DNA Barcode Identification Method of Saiga tatarica and Its Adulterants Based on CO Ⅰ Gene / 中国药房

OBJECTIVE:To investigate the feasibility of molecule identication of Saiga tatarica and its adulterants by using cytochrome C oxidase subunit Ⅰ (CO Ⅰ) gene.METHODS:A total of 7 horns and incomplete horns were collected from 4 areas.The extraction effect of DNA from bone plug and stratum corneum were investigated;PCR technology was used to amplify CO Ⅰ gene of samples using universal primer LCO Ⅰ 490,HCO2198;after gel electrophoresis,purification (750 bp strip) and sequencing,using CO Ⅰ gene as barcode alignment sequence,online comparison was conducted by using Blast software of NCBI database to determine specific species.RESULTS:The extraction of DNA from stratum corneum was better (DNA concentration was 15.7-22.6 ng/μL,the absorbance of 260 nm/280 nm was 1.73-4.72).Online comparison showed that the similarity of CO Ⅰ gene in all sampies reached 99％,mainly from the horns of saiga antelope,Tibetan antelope,gazelle,sheep and goats.CONCLUSIONS:DNA barcode technology based on CO Ⅰ gene can be used for the identification of S.tatarica and its adulterants.The technology can provide an accurate and objective method for the identification of horn medicinal materials.

"A case of lymphocutaneous sporotrichosis caused by Sporothrix schenckii sensu stricto finally diagnosed by molecular identification based on ""ITS"" and ""CAL"" coding regions in combination with phenotypic analysis" / 中华皮肤科杂志

A 54-year-old female farmer presented with a pea-sized red nodule on the left upper limb near the wrist for 15 days,which occurred after trauma,gradually became swollen and ruptured,and developed into multiple nodules arranged in a chain in 30 days.Skin examination revealed multiple hard purple-red nodules arranged in a line on her left upper limb,some of which were ruptured with a small amount of purulent exndate.Histopathological examination further revealed that the focus of infection manifested as pyogenic granuloma-like inflammation mainly infiltrated with mixed inflammatory cells.Periodic acid Schiff (PAS) staining showed no fungal structures,including fungal spores,hyphae and asteroid body.The biopsy tissue culture yielded the fungus.According to the morphological analysis of the cultures and results of molecular identification based on internal transcribed spacer (ITS) and calmodulin (CAL) coding regions,this case was finally diagnosed as lymphocutaneous sporotrichosis caused by Sporothrix schenckii sensu stricto.The patient was treated with oral potassium iodide 10％ solution in a dose of 10 ml thrice a day.After 2-month treatment,the patient felt that the lesions were obviously improved,but afterwards she was lost to follow-up.This research report suggests that phenotypic analysis combined with ITS/CAL-based molecular identification can accurately identify Sporothrix schenckii complex at the species level.

Expression, purification of annexin A4-EGFP fusion protein and preliminary study on its apoptosis detection ability / 药学学报

Annexin is a protein of evolutionarily conserved polygene family that binds to cell membrane phosphatidylserine (PS). PS is closely related to many diseases with a potential as a new drug target. Annexin has a good value in drug discovery and new drug development. Annexin A4 is a member of the annexins family. Annexin A4 involves in a number of cellular functions, such as exocytosis and coagulation. These functions are related to binding of annexin to acidic phospholipids. However, the detail function(s) of annexin A4 has not been fully uncovered. Production of annexin A4 in large quantity is prerequisite for indepth investigation of the structure-function relationship of annexin A4. Human annexin A4 was originally purified from the natural resource at a low yield due to the complex procedure. In the present study, annexin A4 was expressed in a prokaryotic system with a high yield of soluble protein. The plasmid pET28a-annexin A4-EGFP was constructed for the expression. Recombinant annexin A4-EGFP was purified using two methods. Affinity chromatography approach gave a protein yield at purity of 80%. While, the membrane absorption method produced the protein with the purity over 90%. Flow cytometric analysis showed that the annexin A4-EGFP fusion protein could recognize and bind to the apoptotic cells with an affinity PS at 79.58±11.68 nmol·L-1, which is at the same order of magnitude as A5-EGFP. We successfully achieved the efficient expression of annexin A4-EGFP in prokaryotic system, and provided an easy and convenient method for purifying a large amount of annexin A4-EGFP with a high purity. This study has laid a solid foundation for our study of the function of annexin A4 in the future.

Genomic characteristics of 2 strains of influenza A (H9N2) virus isolated from human infection cases in Anhui province / 中华流行病学杂志

Objective To understand genomic characteristics of 2 strains of influenza A (H9N2) virus isolated from human infection cases in Anhui province in 2015.Methods Two human infection with H9N2 virus were confirmed by national influenza surveillance laboratory network in Anhui through viral isolation in April and September,2015,respectively.The full genomic sequences of the two viral isolates were analyzed in this study by using molecular bioinformatics software Mega 6.0.Results Human infection with H9N2 virus was first reported in Anhui province.The analysis of genomic sequence showed that the HA and NA genes of the two H9N2 isolates belonged to A/Chicken/ Shanghai/F/98(H9N2)-like lineage,and shared high identity with H9N2 virus circulating in poultry in 2013.The PB2 and MP genes belonged to the A/quail/Hong Kong/G 1/97-like lineage,and shared high homology with H7N9,H10N8 or H6N2 viruses.The amino acid sequence alignment results showed that several mutations for human infection tropism presented in the two virus strains,including Q226L,H183N and E190T in HA;S31N in M2;63-65 deletion in NA.In addition,the H9N2 influenza virus strains possessed the PSRSSR\GL motif in HA.Meanwhile several human-like signatures,including PA-100A,PA-356R and PA-409N were also found in the two virus strains.Conclusion The H9N2 viruses isolated from human infection cases in Anhui province belonged to a reassortant virus originated from different lineage H9N2 avian influenza virus.The virus has possessed several human susceptibility locus.

Genomic characteristics of 2 strains of influenza A (H9N2) virus isolated from human infection cases in Anhui province / 中华流行病学杂志

Objective To understand genomic characteristics of 2 strains of influenza A (H9N2) virus isolated from human infection cases in Anhui province in 2015.Methods Two human infection with H9N2 virus were confirmed by national influenza surveillance laboratory network in Anhui through viral isolation in April and September,2015,respectively.The full genomic sequences of the two viral isolates were analyzed in this study by using molecular bioinformatics software Mega 6.0.Results Human infection with H9N2 virus was first reported in Anhui province.The analysis of genomic sequence showed that the HA and NA genes of the two H9N2 isolates belonged to A/Chicken/ Shanghai/F/98(H9N2)-like lineage,and shared high identity with H9N2 virus circulating in poultry in 2013.The PB2 and MP genes belonged to the A/quail/Hong Kong/G 1/97-like lineage,and shared high homology with H7N9,H10N8 or H6N2 viruses.The amino acid sequence alignment results showed that several mutations for human infection tropism presented in the two virus strains,including Q226L,H183N and E190T in HA;S31N in M2;63-65 deletion in NA.In addition,the H9N2 influenza virus strains possessed the PSRSSR\GL motif in HA.Meanwhile several human-like signatures,including PA-100A,PA-356R and PA-409N were also found in the two virus strains.Conclusion The H9N2 viruses isolated from human infection cases in Anhui province belonged to a reassortant virus originated from different lineage H9N2 avian influenza virus.The virus has possessed several human susceptibility locus.

In vitro validation of self designed “universal human Influenza A siRNA”.

The genomic variability of Influenza A virus (IAV) makes it difficult for the existing vaccines or anti-influenza drugs to control. The siRNA targeting viral gene induces RNAi mechanism in the host and silent the gene by cleaving mRNA. In this study, we developed an universal siRNA and validated its efficiency in vitro. The siRNA was designed rationally, targeting the most conserved region (delineated with the help of multiple sequence alignment) of M gene of IAV strains. Three level screening method was adopted, and the most efficient one was selected on the basis of its unique position in the conserved region. The siRNA efficacy was confirmed in vitro with the Madin Darby Canine Kidney (MDCK) cell line for IAV propagation using two clinical isolates i.e., Influenza A/H3N2 and Influenza A/pdmH1N1. Of the total 168 strains worldwide and 33 strains from India, 97 bp long (position 137-233) conserved region was identified. The longest ORF of matrix gene was targeted by the selected siRNA, which showed 73.6% inhibition in replication of Influenza A/pdmH1N1 and 62.1% inhibition in replication of Influenza A/H3N2 at 48 h post infection on MDCK cell line. This study provides a basis for the development of siRNA which can be used as universal anti-IAV therapeutic agent.

In silico designing of siRNA targeting PB 1 gene of Influenza A virus and in vitro validation.

Genomic variability makes Influenza A virus (IAV) ‘the least susceptible’ to existing vaccines or anti-influenza drugs. siRNA targeting viral gene silents the gene by cleaving mRNA. Present study aimed to develop siRNA targeting polymerase basic 1 (PB1) gene and to validate its efficiency in vitro. siRNA was designed rationally, targeting the most conserved region of PB1 gene of IAV strains. Total 147 strains worldwide and 42 Indian strains, when aligned, showed seven sets of conserved regions (> 30 bp stretch and < 5% mismatches). To choose the most efficient siRNA, three levels screening method was developed. Finally one pair of siRNA was chosen due to its unique position in conserved region. siRNA efficacy was confirmed in vitro on Madin Darby Canine Kidney (MDCK) cell line propagating two clinical isolates i.e. Influenza A/H3N2 [A/India/LKO864/ 2011(H3N2)] and Influenza A/pandemicH1N1 [A/India/LKO2151/2012(H1N1)]. The longest ORF was targeted by the selected siRNA, which showed 57 % inhibition in replication of Influenza A/pdmH1N1 and 60.6 % inhibition in replication of Influenza A/H3N2 at 72 hpi and 48 hpi respectively on MDCK cell line. This study shows that siRNA targeting PB1 may be moderately effective in controlling IAV replication so can be used as anti-IAV therapeutic agent.

A Primer for Disease Gene Prioritization Using Next-Generation Sequencing Data

High-throughput next-generation sequencing (NGS) technology produces a tremendous amount of raw sequence data. The challenges for researchers are to process the raw data, to map the sequences to genome, to discover variants that are different from the reference genome, and to prioritize/rank the variants for the question of interest. The recent development of many computational algorithms and programs has vastly improved the ability to translate sequence data into valuable information for disease gene identification. However, the NGS data analysis is complex and could be overwhelming for researchers who are not familiar with the process. Here, we outline the analysis pipeline and describe some of the most commonly used principles and tools for analyzing NGS data for disease gene identification.

Genetic analysis of HA gene of pandemic H1N1 2009 influenza viruses circulating in India.

The H1N1 2009 influenza pandemic took the health care workers by surprise in spite of warning about influenza pandemic. Influenza A virus has the ability to overcome immunity from previous infections through the acquisition of genetic changes by shift or drift. Thus, understanding the evolution of the viruses in human is important for the surveillance and the selection of vaccine strains. A total of 23 pandemic A/H1N1 2009 viral HA gene sequences were downloaded from NCBI submitted during March and May 2010 by NIV and were analysed. Along with that the vaccine strain A/California/07/2009 was also downloaded from NCBI. All the sequences were used to analyse the evolution of the haemagglutinin (HA) by phylogenetic analysis. The HA gene could be divided into four groups with shift from 1 to lV revealing that the HA genes of the influenza A viruses evolved in a sequential way, in comparison to vaccine strain A/California/07/2009. Amino acid sequence analysis of the HA genes of the A/H1N1 2009 isolates, revealed mutations at positions 100, 220 and additional mutations in different positions 114, 171, 179, 190, 208, 219, 222, 239, 240, 247, 251, 260 and 285 .The mutations identified showed the adaptation of the new virus to the host that could lead to genetic changes inherent to the virus resulting in a reassortant which could be catastrophic, hence continuous monitoring of strains is mandatory.