Molecular Identification of Gallibacterium from Hen and Analysis of Its 16S rRNA Gene

Two strains of Gallibacterium, isolated from one laying hen flock in Zhengzhou City of Henan Province, were identified by the morphological observation, genus -specific PCR, and analysis of 16S rRNA gene, which was used to generate the phylogenetic tree, with the 21 members of the 12 genera belonging to Pasteurellaceae to analyze the homology. Two strains were named Yu-ZZ-HL-I-SLG and Yu-ZZ-HL-II-GZ. The comparative result of the 16S rDNA sequence shows that the 2 isolated strains are identical in sequence; the highest identity （99.9%） was observed between the isolated strain and one of the strains of Gallibacterium anatis （AF22.8002）, the homologies between the isolated strain and 3 strains of gallibacterium accessed in NCBI （AF22.8016, Gallibacterium genomosp. 1, AF228017, Gallibacterium genomosp. 2, AF22.8018, Gal- libacterium genomosp. 1 ） were above 97.1%, higher than that of the isolated strain and the other strains of the other 11 genera which were between 90.7% -93.2%. It can be seen from the phylogenetic tree that the 2 isolated strains and the other 4 strain of gallibacterium fell into the same branch, furthermore the 2 isolated strains and the strain of Gallibacterium anatis locate in an internal branch, indicating that the 2 isolated strains belong to Gallibacterium anatis.

Sequence and phylogenetic analysis of chicken reoviruses in China

supported by the China Animal Disease Prevention and Control Center;the China Agriculture Research System Poultry-Related Science and Technology Innovation Team of Peking, China （CARS-PSTP）

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 Variation Analyses of nsp2 Gene of PRRSV in Ningxia Hui Autonomous Region of China

To gain a better understanding of the genetic diversity and evolution of PRRSV in the Ningxia Hui Nationality Autonomous Region(Ningxia) of China,the nsp2 genes from a series of PRRSV strains collected from the region in 2007 were partially sequenced. These sequences were then analyzed along with the classical strain(ch-1a) and two other epidemic strains SD(3) and SD2006. Comparison of the nucleotide sequence with ch-1a indicated that nsp2 genes of seventeen Ningxia isolates(NX strain) have deletions of 87 nucleotides. Sequence analysis indicated that homology between the Ningxia strain and ch-1a was 60.3%-79.9% in the nucleotide sequence,and homology between the NX strains and SD strains was 80.3%-98.8% in the nucleotide sequence. The nsp2 genes of the seventeen isolates had 74.9%-100% nucleotide sequence identities with each other. This study was undertaken to assess the regional variation of prevalent PRRSV and to establish a sequence database for PRRSV molecular epidemiological studies.

Molecular Characterization of Duck Hepatitis B Virus Isolated from Hubei Brown Ducks

The objective of this study was to characterize the genome structure of duck hepatitis B virus （DHBV） isolated from Hubei brown ducks. The natural carrier rate of DHBV in adult ducks from Hubei area was investigated and the DHBV DNA-positive serum screened out. The complete genome of a DHBV strain was amplified by polymerase chain reaction （PCR） and cloned into T vector and sequenced. The results showed that the carrier rate of DHBV in Hubei brown ducks was 10 % This strain （GenBank accession number DQ276978） had a genome of 3024 nucleotides with three overlapping open reading frames encoding the surface, core and polymerase proteins respectively. Comparison of the strain with 17 DHBV strains registered in GenBank revealed a homology from 89.3 % to 93.5 % at the nucleotide level. The sequences of the structural and functional domains of these proteins were highly conserved. The strain was found to share more signature amino acids in the polymerase genes with the ＂Chinese＂ DHBV strains than those of the ＂Western＂ country strains. This finding was also corroborated by a phylogenetic tree analysis. Therefore, the DQ276978 might belong to a subtype of the Chinese DHBV strains.

Molecular Clone, Expression, and Prediction of Construction and Function to Key Genes of Interleukin Family of Porcine

This research was to clone, express, and analyze the structure and function of major molecules of porcine interleukin family. Genes of porcine interleukin family were cloned by RT-PCR from stimulated porcine PBMC by LPS and PHA, and then expressed in E. coli, and the structure and function of these molecules were predicted by ExPASY. The results showed that genes of IL-4, IL-6, and IL-18 were successfully cloned and expressed. Furthermore, the expression products of recombinant IL-4 and IL-6 both have multiple biological activities. By analyzing these genes with the NCBI/GenBank data, the homologies of the nucleotide acid sequence are 99.25, 99.21, and 100%, respectively, and have great species differences when compared with other animal species. The results of the prediction showed that all these molecules contain several phosphorylation, glycosylation, protein kinase, and signal transduction bonding sites in secondary structure, and all are compact globularity protein in space configuration. These characteristics of structure are the basis for their multiple biological functions. The genes, structure and function of key molecular of porcine interleukin family were successfully cloned, expressed, and analyzed in this paper.

Molecular characteristics of the omp A gene of serotype B Chlamydia trachomatis in Qinghai Tibetan primary school students

To study the molecular characteristics of Chlamydia trachomatis, the major outer membrane protein gene(omp A) of C. trachomatis from primary school students with trachoma residing in the Qinghai Tibetan area was sequenced and compared with the same serotype in Gen Bank. In Jianshetang Primary School and Galeng Central Primary School in the Galeng Tibetan Township of Qinghai Haidong Sala Autonomous County, scraped samples were collected from the upper tarsal conjunctiva and lower conjunctival sac of both eyes of 45 students with trachoma, stored at 4°C, and transported to Beijing Tongren Hospital by air within 24 h. The samples were screened for C. trachomatis by real-time PCR. The omp A gene from the C. trachomatis-positive samples was amplified by nested PCR. The serotype was confirmed by National Center for Biotechnology Information(NCBI) BLAST search and homology analysis. The entire omp A gene sequence was compared with the corresponding gene sequences of serotype B strains available in Gen Bank. Of the 45 students aged 6–13 years with trachoma, 26 C. trachomatis-positive students were identified by the initial real-time PCR screening(average age,(9.09±1.63) years; sex ratio, 1.0), accounting for 57.78%(26/45). The cycle threshold values for real-time PCR were 16.79–37.77. Half(13/26) of C. trachomatis-positive students had a bacterial copy number of >105. The compliance rate of the omp A gene sequences with the C. trachomatis serotype B strains in Gen Bank was up to 99%. Two novel genetic mutations were found when the omp A gene was compared with those of the 11 serotype B strains in Gen Bank. The two non-synonymous mutations were located at(i) position 271 in the second constant domain, an adenine(A) to guanine(G) substitution(ACT?GCT), changing the amino acid at position 91 from threonine to alanine(Thr?Ala) in all 26 strains; and(ii) position 887 in the fourth variable domain, a cytosine(C) to thymine(T) substitution(GCA?GTA), changing the amino acid at residue 296 from alanine to valine(Ala?Val) in four of the 26 strains. Six mutations were identified relative to ATCC VR-573. The strains could be divided into two gene clusters according to the mutation at nucleotide position 887: CQZ-1(China Qinghai Tibetan-1) and CQZ-2(China Qinghai Tibetan-2). We thus detected two novel serotype B mutant strains of C. trachomatis among study subjects with trachoma.

The complete genome of Enterovirus 71 China strain

Five overlapping clones covering the full genome of Enterovirus71 China strain SHZH98 were obtained and then the sequences were determined by the chain termination method. It showed that the full length of EV71 SHZH98 genome (not including Poly A tail) is 7408 bp. There are some diversities on the lengths and sequences of 5′ UTR and 3′ UTR between SHZH98 and the other EV71 strains. In P1 capsid region, which is closely associated with viral immunogenicity, EV71 strain SHZH98 shares the highest homology with Taiwan strains; but in P2 and P3 non-structural gene regions there are higher identities with Coxsakievirus A16 and EV71 strains MS, BrCr than with Taiwan strains. Phylogenetic tree constructed by structural gene region indicates that China strain SHZH98 has a closer relationship with Taiwan strains, however, in the non-coding region it has a closer relationship with Coxsakievirus A16, EV71 strains MS and BrCr. EV71 China strain was analyzed at the molecular level. The results will contribute to the basic study on enteroviruses and the EV71 prevention in China.