Sequence Analysis of HA Genes from Three H9N2 Subtype Avian Influenza Viruses

[ Objective] The study aimed to understand the genetic characters of H9N2 subtype avian influenza viruses isolated in Belling area. [ Method] HA genes of three H9N2 subtype avian influenza viruses A/Chicken/Beijing/xu/00, A/Chicken/Beijing/bei/00 and A/Chicken/Beijing/ liu/00 were amplified by RT-PCR and then sequenced. [ Result] The results of phylogenetic analysis showed that A/Chicken/Beijing/xu/00, A/ Chicken/Beijing/bei/00 and A/Chicken/Beijing/liu/00 shared the nucleotide homologies of 84.8% （ Dk/HK/Y439/97 ） -98.0% （ Ck/GX17/00 ）, 85.1% （Dk/HK/Y439/97） - 99.1% （ Ck/GXl 7/00）, 90.7% （ Ck/BJ/3/01 ） - 99.1% （Ck/GX17/00） with the isolates from Hongkong and other are- as of Chinese Mainland respectively. At the same time, the analysis of amino acid indicated that the three isolates belonged to low pathogenic H9N2 isolates of avian origin. The 226^th amino acid of them were L （ Leu）, suggesting their high binding affinity to human cells. There were seven glyco- sylation sites in HA protein, five from HA1 and two from HA2. [ Cenclusien] By analysis at molecular level, it could be concluded that A/Chicken/ Beijing/xu/00, A/Chicken/Beijing/bei/00 and A/Chicken/Beijing/liu/00 were low pathogenic H9N2 isolates of avian origin.

Cloning and Sequence Analysis of Sheeppox Virus RPO30 Gene

[Objective] The study aimed to clone RPO30 gene from Sheeppox virus （SPPV） and predict the structure and function of the sequence. [Method] RPO30 gene of SPPV was cloned with PCR, linked into pMD18-T simple vector and then transformed into E. coli DH5a. In blue-white screen, the white colonies were selected to prepare plasmids. The positive plasmids were selected by double digestion and PCR, and then sequenced. Finally, the structure and function of the sequence obtained were predicted by bioinformatics methods. [Results] The RPO30 gene was successfully obtained; its ORF was 585 bp, encoding 193 amino acids and containing a recognition site for Hind III. Moreover, the SPPV RPO30 gene shared different homologies with the RPO30 gene sequences of other pox virus strains from GenBank database. Further analysis by biological software showed that in RPO30 protein, amino acids 4-12, 18-26, 50- 61, 68- 92 and 176-190 had a high possibility to form the active center, and acting to these regions was likely to inactivate the enzyme encoded by the sequence, thus to inhibit viral replication efficiently. [Conclusion] This study will lay foundation for further study on the structure and function of RPO30.

Cloning and Sequence Analysis of E Gene from Chicken Flavivirus Isolate CJD05

[ Objective] This experiment aimed to find out the origin and genetic evolution relationship of chicken flavivirus （CFV） CJD05 strain in Fujian Province. [Method] A pair of primers were designed and synthesized according to the sequences of E gene from Duck flavivirus （DFV） iso- late BYD-1. E gene of CFV isolate CJD05 was specially amplified and its sequences were analyzed. [Result] The target bar which was cloned from CFV isolate C, JD05 was I 503 bp. Homology analysis was conducted to compare E gene nucleotide sequence of CFV isolate CJD05 with DFV iso- late BYD-I and goose flavivirus （GFV） isolate JS804. Results indicated that isolate nuclectide homologies were 99.2% and 99.3%, and amino acid homologies were 99.0% and 98.6% respectively. [Conclusion] CFV isolate C, JD05, DFV isolate BYD-1 and GFV isolate JS804 were highly homologous. The homology of CFV isolate CJD05 with Tembusu virus （TMUV） was higher than with other arthropod-borne flaviviruses.

Cloning and sequence analysis of US1 gene in duck enteritis virus

In this paper,a 1,860 bp sequence in IRs region of duck enteritis virus(DEV) was amplified by single oligonucleotide nested PCR with a single primer designed according to partial sequence of US1 and then a pair of primers designed according to the 3' UTR of US8 gene and 5' end of the new getting sequence were used to amplify a 2,426 bp sequence toward the TRs region.Sequence analysis revealed that the both sequences contained an identical 990 bp open reading frame of DEV US1 gene.The two ORFs were in opposite transcription orientation.Sequence comparison of the nucleotide sequence and the deduced amino acid sequence of US1 gene showed relatively high identity to Mardivirus.Phylogenetic tree analysis showed that the eleven herpesviruses viruses were classified into three groups,and the duck enteritis virus was most closely related to Mardivirus.

Sequence analysis of human papillomavirus type 16 E6E7 gene from cervical carcinoma biopsies of Chinese patients in Shandong province

Objective To study the structure specificity of HPV16E6E7 gene from cervical carcinoma biopsies of patients in Shandong province. Methods The tissue DNAs were extracted from cervical carcinoma biopsies of 14 patients of Chinese from Shandong province. By PCR method using HPV multiple primers, the HPV types were identified in cervical carcinoma tissues, Using the tissue DNA of the 2 cases with the infection of HPV16 type only as templates, HPV16E6E7 gene was amplified by PCR respectively, then inserted the HPV16E6E7 gene into pALTER I vector, and obtained the recombinant plasmid pALTER HPV16E6E7. The constructs were sequenced using Sanger method, and then compared with the sequence of HPV16E6E7 gene of the prototype. Results Sequencing results showed that HPV16E6E7 gene in the 2 cases had sequence diversity from that of the prototype, and a total of five nucleotide exchanges were detected, four of these led to amino acid exchanges. Conclusion There are structure differences between the HPV16E6E7 of Chinese and that of the prototype.

Cloning and sequence analysis of E2 gene of bovine viral diarrhea virus HB-DCZ strain in Hebei province of China

The objective of this paper was to analyze the E2 genetic characterization of HB-DCZ strain of Bovine viral diaxrhca Virus （BVDV） which wcrc amplified by RT-PCR and isolated from China. The product of PCK was cloned into pMD18-T vector, and then transfected Escherichia Coli JMI00. The recombinant plasmids were amplified by PCR and were sequenced. From the nucleotide sequence of the amplified products, phylogenetie analyses were performed and genotypes or subgenotypes were identified. The results indicated that the E2 gene fragment of HB-DCZ strain contained 1277bp nucleotides, and had 89.4%, 70.7%, 97.6%, 68.9%, 67.2% sequence similarity with Osloss, OregonC24V, Changchun184, ZM195, NADL, respectively. In conclusion, HB-DCZ strain is closely related to BVDV Osloss, Changchun184, and belongs to subgenotype lb.

Cloning and Sequence Analysis of the gp41 Gene of Clanis bilineata Nuclear Polyhedrosis Virus

Clanis bilineata Nucleo Polyhedro Virus （CbNPV） was purified from Clanis bilineata larva. To obtain the molecular information of the virus, the genomic DNA of CbNPV was extracted, and a DNA fragment library of the virus was constructed using shotgun. The positive clones were then sequenced and analyzed. An open-reading frame （ORF） that has high identity with the gp41 gene of most NPVs was found in the library. The gp41 gene of CbNPV is 933 base pair long and encodes a protein of 310 amino acids. The result of the amino acid sequence analysis showed that the CbNPV gp41 has 53-61 and 56-73% identities with Group 1 and II NPVs gp41 proteins, respectively. The result indicates that the isolated CbNPV is a novel baculovirus, and the CbNPV shares a much closer relationship Ⅱ NPVs.

Cloning and Sequence Analysis of Envelope Glycoprotein E1 Gene of Rubella Virus, JR23 Strain

To construct an expression vector containing the E1 glycoprotein gene of rubella virus for the study on the effect of mutation of the E1 gene glycoprotein and the analysis of phylogenetic differences of sequences, the gene encoding the E1 envelope glycoprotein was amplified from rubella virus, Jinan strain JR23, by RT-PCR and ligated into PMD-18T vector. The clones that carried the E1 gene were identified after amp r selection and analysis of restriction enzyme digestion. After sequencing this gene was analyzed by Danstar and Winstar programs, and the map of phylogenetic tree was drawn. The clone of E1 glycoprotein was thus constructed. It was found that the sequence differences between JR23 strain and the TCRB strain from Japan and those between JR23 strain and Thomas strain of England were rather small with difference values of 0.9% and 1.2% respectively. Yet those between JR23 strain and BRD2 strain from Beijing and those between JR23 strain and XG379 strain from Hong Kong were comparatively larger with difference values of 7.6% and 7.3% respectively. The sequence of JR23 strain with other strains was less than 3% except the NC strain (3.7%). It concludes that the construction of E1 glycoprotein gene offers an approach to study the relationship between structures and functions of E1 gene and its gene products. In the phylogenetic tree, it shows that there are significant differences in the sequences of rubella virus isolated in China, and this might be helpful to develop an effective subunit vaccine.

Analysis on Heredity and Variation of the ORF_5 Gene of Prevalence Strains Porcine Reproductive and Respiratory Syndrome Virus

[ Objective] The aim was to analyze the reason and epidemic trend of PRRSV, and provide theoretical basis for preventing and controlling PRRS. [Methed]According to the sequence of ATCC VR-2332 strain porcine reproductive and respiratory syndrome virus published by the GenBank, the primers were designed and synthesized. ORF5 gene sequences of seven prevalence strains were amplified by RT-PCR. The sequences of ORF5 genes were analyzed by DNAStar and compared with those of ATCC VR-2332, CH-1 a, B J-4, LV-M96262 and MLV vaccine strains, phylogenetic tree among isolates was analyzed. [Result] Analysis of nucleotide sequence showed that the homology was 88.1% - 98.8%, 89.9% -95.2%, 85.6% -98.7% between ORF5 genes of seven prevalence strains and VR-2332, CH-1a, BJ-4, and the homology was 54.7% -56.9% between ORF5 genes and LV. Analysis of amino acid sequence showed that the homology was 88.1% -96.8%, 88.1% - 94.5%, 86.1% -96.5% between ORF5 genes of seven prevalence strains and VR-2332, CH-1a, bBJ-4, the homology was 54.7% -56.2% between the ORF5 genes and LV.[ Conclusion] The variation of prevalence strains was great in the ORF5 gene region, the homology of ORF5 gene sequence was higher and genetic relationship was nearer during prevalence strains in the same region, or was far in different regions.

E Sequence Analysis of Persistently Infected Mutant Japanese Encephalitis Virus Strains

Summary： A persistent infection model was established after human hepatoma cells infected by Japanese encephalitis viruses were subcultured for several times. Viral titers of mutant viruses in persistently infected cells were examined by plaque methods using BHK cells. Nucleotides of the E coding region of two wild and two mutant viruses were amplified by RT-PCR. PCR products were sequenced by ABI-PRSM^TM310 sequencing system. Compared to JaGAr-01 wild strains, four amino acids were replaced （E61Tyr→Asp, E219His→Tyr, E384Val→Glu, E418Pro→Ala） in the E sequence of JaGAr-01 persistently-infected mutant strains. Eleven amino acid replacement （E51Arg →Ser, E61Tyr→Asp, E83Lys→Glu, E123Ser→Arg, E209Arg→Lys, E227Pro→Ser, E276Asp→er, E290Arg→Lys, E387Lys→Arg, E418Leu→Pro, E454Arg→Gly） was also noted when we compared the E sequence between persistently infected Nakayama and its wild strains. A lot of similarities of amino acid sequence between mutant strains JaGAr-01 and Nakayama were also noted. It was concluded that geno-variation existed in E region of mutant viruses and the mutant protein encoded by E region, especially the mutation of E61 （Tyr→Asp） may contribute to the maintenance of the persistent infection of Japanese encephalitis virus.

HA Gene Variation Analysis of H9N2 Sub-type Avian Influenza Virus from Three Strains in Different Times

HA Gene of H9N2. sub-type avian influenza virus from three strains in different times was amplified, purified and then sequenced. The results of its sequence analysis showed that the whole length of the amplified HA Gene was 1 683 bp, encoding 560 amino acids. The amino acid sequence of three virulent strains at cleavage site was R-S-S-R, which was low-pathogenicity strain. According to the amino acid sequence of the isolated strains, there were 7 potential glycosylation sites, and the receptor-binding site was the specific sequence of the avian-derived influenza virus. Amino acids on the left edge of receptor-binding site were all NGQQG, while amino acids on the right edge of receptor-binding site were GTSKA. From the comparative sequence analysis of HA Gene from some referenced strains, the results indicated that nucleotide and amino acid homology between isolated strains and referenced strains was higher. Evolutionary tree analysis showed that three strains were all Eurasian species, and there was a close relationship with the representative strains of A / duck / Hong Kong/Y280/97.

Genetic Diversity of Chinese Soybean mosaic virus Strains and Their Relationships with Other Plant Potyviruses Based on P3 Gene Sequences

Soybean mosaic virus （SMV）, a member of the genus Potyvirus, is a major pathogen of soybean plants in China, and 16 SMV strains have been identified nationwide based on a former detailed SMV classification system. As the P3 gene is thought to be involved in viral replication, systemic infection, pathogenicity, and overcoming resistance, knowledge of the P3 gene sequences of SMV and other potyviruses would be useful in efforts to know the genetic relationships among them and control the disease. P3 gene sequences were obtained from representative isolates of the above-mentioned 16 SMV strains and were compared with other SMV strains and 16 Potyvirus species from the National Center for Biotechnology GenBank database. The P3 genes from the 16 SMV isolates are composed of 1041 nucleotides, encoding 347 amino acids, and share 90.7-100% nucleotide （NT） sequence identities and 95.1-100% amino acid （AA） sequence identities. The P3 coding regions of the 16 SMV isolates share high identities （92.4-98.9% NT and 96.0-100% AA） with the reported Korean isolates, followed by the USA isolates （88.5-97.9% NT and 91.4-98.6% AA）, and share low identities （80.5-85.2% NT and 82.1-84.7% AA） with the reported HZ 1 and P isolates from Pinellia ternata. The sequence identities of the P3 genes between SMV and the 16 potyviruses varied from 44.4 to 81.9% in the NT sequences and from 21.4 to 85.3% in the AA sequences, respectively. Among them, SMV was closely related to Watermelon mosaic virus （WMV）, with 76.0-81.9% NT and 77.5-85.3% AA identities. In addition, the SMV isolates and potyvirus species were clustered into six distinct groups. All the SMV strains isolated from soybean were clustered in Group I, and the remaining species were clustered in other groups. A multiple sequence alignment analysis of the C-terminal regions indicated that the P3 genes within a species were highly conserved, whereas those among species were relatively variable.

Genetic Analysis of the Entire Genome of a A/duck/Shanghai/Y20/2006 (H4N6) Avian Influenza Virus

[ Objective] This paper aimed to investigate the origin, characteristics and molecular evolution of duck derived H4N6 subtype avian influ- enza virus （DK/SH/Y20/06） and enrich the epidemiologic data of the waterfowl origin AIV. [Method] The entire genome of DK/SH/Y20/06 was amplified and subjected to genome sequencing. The molecular software was used for sequence analysis and phylogenetic tree construction of DK/ SH/Y20/06 with some other reference sequences in GenBank. [Result] The results indicated that the amino acid sequence adjacent to HA cleav- age site was PEKASR ↓ GLF, which was the typical characteristics of the LPAIV. The phylogenetic analysis indicated that the HA gene of the isolate was derived from the Eurasian lineage in the eastern hemisphere. The NA gene was at the same branch with A/rnallard/Yan chen/2005（ H4N6）, sharing 98.3% sequence identity. The PB2, PB1, NP and PA gene of this isolate had genetically close relationships with H6 subtype AIV which is epidemic in China at present. The M gene fell into the same branch with A/environment/Korea/CSM05/2004（ H3N1 ）. The NS segment had the highest similarity with A/wild duck/Korea/YS44/2004（H1N2）. The eight genes were not at the same branch and shared a low similarity with other H4N6 subtype avian influenza viruses isolated in North America. [Condusion] These data showed that DK/SH/Y20/06（H4N6） was possibly a re- combinant virus derived from H4N6 subtype, H6N2, H6N5, H3N1 and H1 N2 subtype AIV by complex gene recombination in duck.

Genome Sequencing and Phylogenetic Analysis of Three Avian Influenza H9N2 Subtypes in Guangxi

Three isolates of H9N2 Avian Influenza viruses (AIV) were isolated from chickens in Guangxi province. Eight pairs of specific primers were designed and synthesized according to the sequences of H9N2 at GenBank. phylogenetic analysis showed a high degree of homology between the Guangxi isolates and isolates from Guangdong and Jiangsu provinces, suggesting that the Guangxi isolates originated from the same source. However, the eight genes of the three isolates from Guangxi were not in the same sublineages in their respective phylogenetic trees, which suggests that they were products of natural reassortment between H9N2 avian influenza viruses from different sublineages. The 9 nucleotides ACAGAGATA which encode amino acids T, G, I were absent between nucleotide 205 and 214 in the open reading frame of the NA gene in the Guangxi isolates. AIV strains that infect human have, in their HA proteins, leucine at position 226. The analysis of deduced amino acid sequence of HA proteins showed that position 226 of these isolates contained glycine instead of leucine, suggesting that these three isolates differ from H9N2 AIV strains isolated from human infections.

Cloning and Sequencing of Kappa Light Chain Gene of a Mouse Monoclonal Antibody Directed Against Potato Virus Y

A cDNA library was constructed in λgt11 vectors, complementary to the mRNA isolated from a mouse hybridoma raised against potato virus Y(PVY). Thirty cDNA clones were selected from the cDNA library by in situ immunohybridization with goat anti-mouse kappa-chain-specific antibody conjugated to alkaline phosphatase, from which one clone, k6, having the largest insert was characterized by sequence analysis. The result shows that the immunoglobulin messenger RNA corresponding to k6 is 956 nucleotides in length excluding the poly(A) region, among which 31 bases code for the 5’ non-coding region, 57 for the leader sequence of the protein, 657 for the mature protein and 211 for the 3’ non-coding region. Comparison of deduced amino acid sequences of the protein and other kappa light chains shows that they share a 100% identity in their constant regions(CL) and 93.7% identity in their variable regions(VL). The kappa light chain encoded by k6 is considered to be specific to PVY since only one type of light chain is expressed in the hybridoma.

Quasispecies structure,cornerstone of hepatitis B virus infection: Mass sequencing approach

Hepatitis B virus(HBV)is a DNA virus with complex replication,and high replication and mutation rates,leading to a heterogeneous viral population.The population is comprised of genomes that are closely related,but not identical;hence,HBV is considered a viral quasispecies.Quasispecies variability may be somewhat limited by the high degree of overlapping between the HBV coding regions,which is especially important in the P and S gene overlapping regions,but is less significant in the X and preCore/Core genes.Despite this restriction,several clinically and pathologically relevant variants have been characterized along the viral genome.Next-generation sequencing(NGS)approaches enable high-throughput analysis of thousands of clonally amplified regions and are powerful tools for characterizing genetic diversity in viral strains.In the present review,we update the information regarding HBV variability and present a summary of the various NGS approaches available for research in this virus.In addition,we provide an analysis of the clinical implications of HBV variants and their study by NGS.

Expression of putative zinc-finger protein lcn61 gene in lymphocystis disease virus China (LCDV-cn) genome

An open reading frame (lcn61) of lymphocystis disease virus China (LCDV-cn), probably responsible for encoding putative zinc-finger proteins was amplified and inserted into pET24a (+) vector. Then it expressed in E. coli BL21 (DE3), and His-tag fusion protein of high yield was obtained. It was found that the fusion protein existed in E. coli mainly as inclusion bodies. The bioinformatics analysis indicates that LCN61 is C2H2 type zinc-finger protein containing four C2H2 zinc-finger motifs. This work provides a theory for functional research of lcn61 gene.

The stability of E protein gene of the Japanese encephalitis live attenuated vaccine virus SA_(14)-14-2

The purpose of this study was to understand the stability and possibility of back mutation of Japanese encephalitis （JE） attenuated vaccine virus strain SA14-14-2 HKs on molecular level. The E genes of the SA14-14-2 HKs vaccine virus and its PHK cells passaged virus （SA14-14-2 HK17 ）, its mouse brain passaged virus （SA14-14-2 SM1 ） were sequenced and compared with the E gene of parental SAI4 virus. The total RNA was extracted from infected Vero cells and amplified by RT-PCR. The RT-PCR products were purified and cloned into T-vector. Positive clones were screened, identified and sequenced. There were twelve nucleotides and eight amino acids substitutions between SA14 parent virus and SA14-34-2 PHKs vaccine virus. The SA14-14-2 PHK17 virus showed two additional mutations （E-331 and E-398） which were not back mutations. Although five additional mutations were found in SA14-34-2 SMt virus, only one （E-307） was back mutation. Genetic characteristics of the attenuated vaccine virus SA14- 34-2 were stable when it was passaged 37 times on PHK cells or one time in mouse brains.

Genotyping and pathotyping of diversified strains of infectious bronchitis viruses circulating in Egypt

AIM: To characterize the circulating infectious bronchitis virus(IBV) strains in Egypt depending on the sequence of the spike-1(S1) gene [hypervariable region-3(HVR-3)] and to study the pathotypic features of these strains.METHODS: In this work, twenty flocks were sampled for IBV detection using RRT-PCR and isolation of IBV in specific pathogen free(SPF) chicks during the period from 2010 to 2015. Partial sequencing and phylogenetic analysis of 400 bp representing the HVR-3 of the S1 gene was conducted. Pathotypic characterization of one selected virus from each group(Egy/Var-Ⅰ, Egy/Var-Ⅱ and classic) was evaluated in one day old SPF chicks. The chicks were divided into 4 groups 10 birds each including the negative control group. Birds were inoculated at one day by intranasal instillation of 105EID50/100 μL of IBV viruses [IBV-EG/1212B-2012(Egy/Var-Ⅱ), IBV/EG/IBV1-2011(Egy/Var-Ⅰ) and IBV-EG/11539F-2011(classic)], while the remaining negative control group was kept uninfected. The birds were observed for clinical signs, gross lesions and virus pathogenicity. The real-time rR TPCR test was performed for virus detection in the tissues. Histopathological examinations were evaluated in both trachea and kidneys.RESULTS: The results revealed that these viruses were separated into two distinct groups; variant(GI-23) and classic(GI-1), where 16 viruses belonged to a variant group, including 2 subdivisions [Egy/Var-Ⅰ(6 isolates) and Egy/Var-Ⅱ(10 isolates)] and 4 viruses clustered to the classic group(Mass-like). IBV isolates in the variant group were grouped with other IBV strains from the Middle East. The variant subgroup(Egy/Var-Ⅰ) was likely resembling the original Egyptian variant strain(Egypt/Beni-Suif/01) and the Israeli strain(IS/1494/2006). The second subgroup(Egy/Var-Ⅱ) included the viruses circulating in the Middle East(Ck/EG/BSU-2 and Ck/EG/BSU-3/2011) and the Israeli strain(IS/885/00). The two variant subgroups(Egy/Var-Ⅰ and Egy/Var-Ⅱ) found to be highly pathogenic to SPF chicks with mortalities up to 50% than those of the classic group which was of low virulence(10% mortality). Pathogenicity indices were 25(Egy/Var-Ⅱ), 24(Egy/Var-Ⅰ) and 8(classic); with clinical scores 3, 2 and 1 respectively.CONCLUSION: These findings indicated that the recent circulating Egyptian IBVs have multiple heterogeneous origins in marked diversifying nature of their spread, with high pathotype in specific pathogen free chicks.

Phylogenetic analysis of avian infectious bronchitis virus isolates from Morocco:a retrospective study(1983 to 2014)

Dear Editor,Infectious bronchitis(IB),one of the most common and difficult poultry diseases,is caused by a gammacoronavirus named infectious bronchitis virus(IBV).IBV frequently causes respiratory and/or renal diseases in chickens and egg production losses in hens.IB has