Complete Genome Sequencing and Genetic Variation Analysis of Two H9N2 Subtype Avian Influenza Virus Strains

[Objective] The study aimed to investigate the genetic variation characters of entire sequences between two H9N2 subtype avian influenza virus strains and other reference strains.[Method] The entire sequences of 8 genes were obtained by using RT-PCR,and these sequences were analyzed with that of six H9N2 subtype avian influenza isolates in homology comparison and genetic evolution relation.[Result] The results showed that the nucleotide sequence of entire gene of the strain shared 91.1%-95.4% homology with other seven reference strains,and PG08 shared the highest homology 91.3% with C/BJ/1/94;ZD06 shared the highest homology 92.3% with D/HK/Y280/97.HA cleavage sites of two H9N2 subtype avian influenza virus isolated strains were PARSSR/GLF,typical of mildly pathogenic avian influenza virus.[Conclusion] Phylogenetic tree for entire gene of eight strains showed that the genetic relationship was the closest between ZD06 and C/Pak/2/99 strains,which belonged to the Eurasian lineage;PG08 shared the highest homology 91.3% with ZD06,it may be the product of gene rearrangements of other sub-lines.

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.

Study on Microsatellite Distribution in Complete Genomes of Tobacco Vein Clearing Virus

MATLAB software and optimal complete subgraph algorithm were used to extract and reveal the microsatellite distribution features in the complete genomes of the tobacco vein clearing virus （NC-003 378.1） from the NCBI database.The results showed that the repetitions number and their location of the N-base group has been extracted and displayed.The largest repetitions of N-base group in the complete genomes of the tobacco vein clearing virus was decreased as the exponential function with the increasing of N.The method used in this study could be applied to the extraction and revealing of the microsatellite distribution features in the complete genomes of other viruses,thereby provided a basis for the research of the structure and the law of function,inheritance and variation by the using of the microsatellite distribution features.

Genetic and pathogenic characterization of new infectious bronchitis virus strains in the GVI-1 and GI-19 lineages isolated in central China

Avian infectious bronchitis(IB)is a highly contagious infectious disease caused by infectious bronchitis virus(IBV),which is prevalent in many countries worldwide and causes serious harm to the poultry industry.At present,many commercial IBV vaccines have been used for the prevention and control of IB;however,IB outbreaks occur frequently.In this study,two new strains of IBV,SX/2106 and SX/2204,were isolated from two flocks which were immunized with IBV H120 vaccine in central China.Phylogenetic and recombination analysis indicated that SX/2106,which was clustered into the GI-19 lineage,may be derived from recombination events of the GI-19 and GI-7 strains and the LDT3-A vaccine.Genetic analysis showed that SX/2204 belongs to the GVI-1 lineage,which may have originated from the recombination of the GI-13 and GVI-1 strains and the H120 vaccine.The virus cross-neutralization test showed that the antigenicity of SX/2106 and SX/2204 was different from H120.Animal experiments found that both SX/2106 and SX/2204 could replicate effectively in the lungs and kidneys of chickens and cause disease and death,and H120 immunization could not provide effective protection against the two IBV isolates.It is noteworthy that the pathogenicity of SX/2204 has significantly increased compared to the GVI-1 strains isolated previously,with a mortality rate up to 60%.Considering the continuous mutation and recombination of the IBV genome to produce new variant strains,it is important to continuously monitor epidemic strains and develop new vaccines for the prevention and control of IBV epidemics.

Complete Genome Sequence Analysis of Duck Circovirus Strains from Cherry Valley Duck

To investigate molecular epidemiology of DuCV in Cherry Valley ducks in China, the complete genomes of six DuCV strains, which were detected from Cherry Valley ducks in China between 2007 and 2008, were sequenced. Sequence and phylogenetic analysis were carried out to compare these six strains with another 27 DuCV strains from Mulard duck, Muscovy duck, Pekin ducks and Mule duck. The analysis showed that the six DuCV strains exhibited typical genetic features of the family of DuCV, such as a stem-loop structure, three major open reading frames (Rep, Cap and ORF3), four intergenic repeats and the conserved motifs for rolling circle replication and for the dNTP binding domain located in the Rep protein. Phylogenetic analysis of the nucleotide sequences of the complete genome and Cap gene of these strains together with those that have been previously published demonstrated two distinct DuCV genotypes. The DuCV strains with complete genomes containing 1988 and 1989 nucleotides clustered in genotype A, whereas the strains with complete genomes containing 1991, 1992, 1995 and 1996 nucleotides lay in genotype B. The six DuCV strains from Cherry Valley ducks were divided into the two groups. The results of the study provides some insight into the variation of DuCVs in Cherry Valley ducks.

Complete Genomic Sequence of Transmissible Gastroenteritis Virus TS and 3' End Sequence Characterization Following Cell Culture

The complete genome sequence of transmissible Gastroenteritis virus (TGEV) strain TS, previously isolated from Gansu province, was cloned and compared with published sequence data from other TGEV strains. Phylogenetic tree analysis based on the amino acid and nucleotide sequences of the S gene showed that the TGEV strains were divided into 3 clusters. TGEV TS showed a close evolutionary relationship to the American Miller cluster but had a 5' non-translated region (NTR) sequence closely related to the American Purdue cluster. Continued culture in different cell types indicated that TGEV TS virulence could be attenuated after fifty passages in Porcine kidney (PK-15) cells, and that the Porcine kidney cell line IB-RS-2 (IBRS) was not suitable for culture of the TGEV strain TS.

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.

Complete genome of Cobetia marina JCM 21022T and phylogenomic analysis of the family Halomonadaceae

Cobetia marina is a model proteobacteria in researches on marine biofouling. Its taxonomic nomenclature has been revised many times over the past few decades. To better understand the role of the surface-associated lifestyle of C. marina and the phylogeny of the family Halomonadaceae, we sequenced the entire genome of C. marina JCM 21022T using single molecule real-time sequencing technology （SMRT） and performed comparative genomics and phylogenomics analyses. The circular chromosome was 4 176 300 bp with an average GC content of 62.44% and contained 3 611 predicted coding sequences, 72 tRNA genes, and 21 rRNA genes. The C. marina JCM 2102U genome contained a set of crucial genes involved in surface colonization processes. The comparative genome analysis indicated the significant differences between C. marina JCM 21022T and Cobetia amphilecti KMM 296 （formerly named C. marina KMM 296） resulted from sequence insertions or deletions and chromosomal recombination. Despite these differences, pan and core genome analysis showed similar gene functions between the two strains. The phylogenomic study of the family Halomonadaceae is relationships were well resolved among every genera Cobetia, Kushneria, Zymobacter, and Halotalea. reported here for the first time. We found that the tested, including Chromohalobacter, Halomonas,

Complete mitochondrial genome of the Thai Red Junglefowl (Gallus gallus) and phylogenetic analysis

DEAR EDITOR,In this study, we sequenced the complete mitochondrial genome （mitogenome） of the Thai Red Junglefowl （RJF; Gallus gallus） using the next-generation sequencing （NGS） platform of the Ion Torrent PGM. Samples were taken from Mae Wang District, Chiang Mai Province, northern Thailand Our data showed the complete mitogenome to be 16 785 bp in length, composed by 13 protein-coding genes, 22 tRNA genes, two rRNA genes, and one control region. The genome nucleotide composition was 30.3% A, 23.7% T, 32.5% C, and 13.5% G, resulting in a high percentage of A＋T （50.4%）. Phylogenetic analysis revealed that the mitogenome belonged to haplogroup X, whereas those of all domestic chickens belong to haplogroups A to G. This newly released mitogenome sequence will advance further evolutionary and population genetics study of the RJF and domestic chicken The availability of the G. gallus mitogenome will also contribute to further conservation genetics research of a unique species, listed as ＇data deficient＇ in Thailand.

A Monophyletic Status of Axis Genus in Subfamily Cervinae Supported by the Complete Mitochondrial Genome of Chinese Hog Deer(Axis porcinus)

Hog deer(Axis porcinus)is a small mammal and listed in the International Union for Conservation of Nature.However,phylogenetic position of hog deer within Axis genus has remained controversial.In the present study,we first assembled complete mitochondrial genome of Chinese hog deer reared in Chengdu Zoo,Sichuan,by the second-generation sequencing technology.This newly assembled mitochondrial genome of hog deer is 16376 bp in length and consists of 13 protein-encoding genes,23 transfer RNA genes and 2 ribosomal RNA genes.Phylogenetic analyses based on complete mitochondrial genome and cytochrome b gene sequences revealed that hog deer is closely clustered together and placed with sister taxon of spotted deer(A.Axis),which therefore supported monophyletic statue of Axis genus.Furthermore,considerable genetic differentiation,up to 139 mutations of complete mitochondrial genome was revealed between geographical populations of hog deer in France and Southeast Asia.However,only six variable sites(nucleotide diversity of 0.00007)and four haplotypes(haplotype diversity of 0.533)were totally detected among ten newly sequenced Chinese hog deer.The results provide a better understanding on the phylogeny of hog deer.

Complete genome sequence of two strawberry vein banding virus isolates from China

It was rarely reported about strawberry vein banding virus(SVBV)genome sequence in China and most countries worldwide.In this work,we determined the complete genome sequences of two SVBV isolates in China,designated SVBV-AH and SVBV-BJ,that were obtained from naturally infected strawberry samples from Anhui province and Beijing city of China,respectively.The complete genomes of SVBV-AH and SVBV-BJ were 7,862 nucleotides(nts)and 7,863 nts long,respectively,and both constituted with seven genes typical of the caulimoviruses.Alignment of complete nucleotide sequences showed that SVBV-AH and SVBV-BJ shared a significant nucleotide sequence identity of 97.7%of each other and had 85.7%and 86.0%sequence identity related to SVBV from the United States(SVBV-US),respectively.Phylogenetic trees,based on the alignment of complete nucleotide sequences and amino acid sequences of Coat Protein(CP),both showed that SVBV-AH and SVBV-BJ clustered into one branch with all the other SVBV isolates,and other species of caulimoviruses clustered into another tree branch.It illustrated that all the SVBV isolates had an extremely high relationship but had a distant relationship with other species of caulimoviruses.We further confirmed that SVBV-AH infectious clone could cause similar symptoms to SVBVinfected in strawberry under natural conditions.Taken together,our study provided valuable information to elucidate the origin and dissemination of SVBV Chinese isolates,meanwhile providing the necessary vector for studying the gene functions of strawberry.

A Comparison of Complete Genome Sequences of a Rabies Virus Chinese Isolate SH06 with the Vaccine Strains

In this study, we determined the complete nucleotide and deduced amino acid sequence of a primary isolate of rabies virus (SH06) obtained from the brain of a rabid dog. The overall length of the genome was 11 924 nucleotides. Comparison of the genomic sequence showed the homology of SH06 at nucleotide level with full-length genomes of reference vaccine strains ranged from 82.2% with the PV strain to 86.9% with the CTN strain. A full-length genome-based phylogenetic analysis was performed with sequences available from GenBank. Phylogenetic analysis of the complete genome sequences indicated that the SH06 exhibited the highest homology with rabies street virus BD06 and CTN vaccine strain originated from China.

Complete genome analysis of bacteriochlorophyll acontaining Roseicitreum antarcticum ZS2-28^(T)reveals its adaptation to Antarctic intertidal environment

Aerobic anoxygenic phototrophic bacteria(AAPB)are photoheterotrophic prokaryotes able to use both light and dissolved organic matter as energy sources.Roseicitreum antarcticum ZS2-28^(T)was isolated from intertidal sediment in the Larsemann Hills,Princess Elizabeth Land,Antarctica,and was able to produce bacteriochlorophyll a.It is the type strain of the sole species within the genus Roseicitreum.The complete genome sequence of the bacterium was determined using Illumina HiSeq X and PacBio RSII systems.The genome of R.antarcticum ZS2-28^(T)was 4253095 bp and consisted of one chromosome and four plasmids.A number of genes related to the bacteriochlorophyll a production,photosynthetic reaction,cold adaptation,salt adaptation,ultra-violet resistance and DNA damage repairing were found in the genome.In addition to genomic islands and typeⅣsecretion systems,genes related to gene transfer agents were detected in the genome of R.antarcticum ZS2-28^(T),suggesting that this bacterium can adapt to its environment by acquiring exogenous nucleic acids.The annotated complete genome sequence provides genetic insights into the environmental adaptation and ecological function of R.antarcticum ZS2-28^(T)in Antarctic coastal area.

Phylogenetic Relationship Analysis of the Complete Genomes of Porcine Circovirus Type 2( PCV2) Strains Isolated from Hainan Province

[ Objective] This study aimed to investigate the molecular characteristics of porcine circovirus type 2 （PCV2） strains isolated from Hainan Province. [ Method] The complete genome of PCV2 was amplified from PMWS-suspected samples by PCR for sequence analysis. [ Result] A total of eight PCV2 strains were isolated and identified. All the eight isolates belonged to genotype PCV2b, among which seven isolates belonged to subgenotype PCV2b-1 C, and one isolate be- longed to subgenotype PCV2b-IA/1B. ORF2 gene of PCV2 isolates from Hainan Province was 705 bp in length, encoding 234 amino acids. Antigenic epitopes of Cap protein exhibited certain changes. Nucleotide sequences and deduced amino acid sequences of ORF2 gene shared 95.3% -99.7% and 93.6% - 100% simi- larities among eight PCV2 isolates from Hainan Province, respectively. Moreover, nucleotide sequences and deduced amino acid sequences of ORF2 gene of PCV2 isolates from Hainan Province shared 91.0% -99.9% and 91.0% -99.6% similarities with other PCV2 strain isolated from China （AY682994, AF381175, JX945577, JX682407, AY180397 ）, respectively; nucleotide sequences and deduced amino acid sequences of ORF2 gene of PCV2 isolates from Hainan Province shared 90.0% - 97.0% and 88.0% -97.9% similarities with PCV2 isolates from other countries （ NC_005148, JQ994268, KJ187306, AF201307, AF454546, AY＂/Sd020）, respectively; nucleotide sequences and deduced amino acid sequences of ORF2 gene of PCV2 isolates from Hainan Province shared 98.2% -100% and 94.9% -100% similarities with vaccine strain SH, respectively; nucleotide sequences and deduced amino acid sequences of ORF2 gene of PCV2 isolates from Hainan Province shared 90.6% -91.7% and 89.7% - 91.0% similarities with vaccine strain LG, respectively. [ Conclusion] This study provided theoretical hasis for the prevention and control of PCV2 and selection of vaccine strains in Hainan Province.

The Full-length Genome Analysis of a Street Rabies Virus Strain Isolated in Yunnan Province of China

The epidemic of rabies has rapidly increased and expanded in Yunnan province in recent years. In order to further analyze and understand the etiological reasons for the rapid expansion of rabies in Yunnan, a strain of rabies virus CYN1009D in Yunnan was isolated, and the complete genomic sequencing was carried out, and the bioimfomative analysis on genes/encoded proteins and phylogeny with reference to sequences in GenBank was performed. The complete genome of CYN1009D was 11923 nt in length and belonged to genotype I. The genes encoding different structural proteins were all conserved in their lengths, in comparison to other strains in China. The amino acid sequence was conserved at different antigen sites of NP, but the variation was detected at the secondary phosphorylation site of position 375; variations were also detected in the phosphorylation sites at positions 63-63 and 162 of PP; the sites playing important roles in virus synthesis, budding and viral morphology in MP were conserved; two glycosylation sites were detected at Asn37 and Ash319 in GP, the neutralizing antigen sites in GP were conserved; the initial amino acid of LP （ML） was different from that of most of the strains in China （MM）; the variations in G-L region in the intergenic region were significant. The phylogenic tree showed that CYN1009D has a closer genetic relationship to the strains in Southeast Asia, indicating that prevention and control on rabies in borderland areas should be reinforced meanwhile efforts are made to control rabies in China.

Molecular Characterization of Banana streak virus Isolate from Musa Acuminata in China

Banana streak virus （BSV）, a member of genus Badnavirus, is a causal agent of banana streak disease throughout the world. The genetic diversity of BSVs from different regions of banana plantations has previously been investigated, but there are relatively few reports of the genetic characteristic of episomal （non-integrated） BSV genomes isolated from China. Here, the complete genome, a total of 7722bp （GenBank accession number DQ092436）, of an isolate of Banana streak virus （BSV） on cultivar Cavendish （BSAcYNV） in Yunnan, China was determined. The genome organises in the typical manner of badnaviruses. The intergenic region of genomic DNA contains a large stem-loop, which may contribute to the ribosome shift into the following open reading frames （ORFs）. The coding region of BSAcYNV consists of three overlapping ORFs, ORF 1 with a non-AUG start codon and ORF2 encoding two small proteins are individually involved in viral movement and ORF3 encodes a polyprotein. Besides the complete genome, a defective genome lacking the whole RNA leader region and a majority of ORF1 and which encompasses 6525bp was also isolated and sequenced from this BSV DNA reservoir in infected banana plants. Sequence analyses showed that BSAcYNV has closest similarity in terms of genome organization and the coding assignments with an BSV isolate from Vietnam （BSAcVNV）. The corresponding coding regions shared identities of 88% and -95% at nucleotide and amino acid levels, respectively. Phylogenetic analysis also indicated BSAcYNV shared the closest geographical evolutionary relationship to BSAcVNV among sequenced banana streak badnaviruses.

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.

Genome Analysis of the Bombyx mori Infectious Flacherie Virus Isolated in China

We reported here the complete genome of the Bombyx mori infectious flacherie virus （BmlFV）, BmlFV-CHN01, isolated in China and compared it with the Japanese strain IFV （BmIFV-JAN） sequence. BmIFV-JAN and BmIFV-CHN01 were 99% identical in nucleotide and amino acid sequences. The amino acid sequences of the three structural proteins （VP1, VP3 and VP4） and L protein were 100% identical between the two strains. Phylogenetic analyses based on conserved domains in RNA-dependent RNA polymerases （RdRps） by the neighbor-joining method showed that these two strains were from the same virus. The gain of the whole genome of the BmIFV isolated in China and its weak mutation character established a great foundation to the study of functional genome and the molecular epidemiology of BmIFV.

Incidence,genomic diversity,and evolution of strawberry mottle virus in China

Strawberry mottle virus(SMoV)is one of the most common viruses infecting strawberries,causing losses to fruit yield and quality.In this study,165 strawberry leaf samples were collected from six provinces of China,46 of which tested positive for SMoV.The complete genome sequences of 11 SMoV isolates were obtained from Liaoning(DGHY3,DGHY16-2,DGHY17,DGHY20-2,DGHY21,DGHY26-2),Shandong(SDHY1,SDHY5,SDHY31-2,SDHY33-2),and Beijing(BJMX7).The RNA1 and RNA2 nucleotide identities between the 11 Chinese isolates were 95.4-99.3%and 96.3-99.6%,respectively,and they shared 78.4-96.6%and 84.8-93.5%identities with the available SMoV isolates in GenBank.Recombination analysis revealed that Chinese isolate SDHY33-2 and Canadian isolates Ontario and Simcoe were recombinants,and recombination events frequently occurred in the 3’UTR of SMoV.Phylogenetic analysis showed that in an RNA1 tree,most Chinese isolates clustered into the same group while isolate DGHY17 clustered into another group together with Czech isolate C and three Canadian isolates.In an RNA2 tree,all Chinese isolates clustered into a single group.The phylogenetic analysis based on nucleotide sequences was consistent with the results based on coat protein(CP)and RNA-dependent RNA polymerase(RdRp).Further evolutionary analysis indicated that negative selection drives SMoV evolution,and gene flow plays a major role in genetic differentiation.Additionally,reassortment and recombination also influence the evolution of SMoV.To our knowledge,this is the first report of the complete genome of SMoV isolates from China and a detailed analysis of the SMoV population structure.

Molecular Characterization and SYBR Green I-Based Quantitative PCR for Duck Hepatitis Virus Type 1

To determine the genomic sequence of a duck hepatitis virus type 1 （DHV-1） strain, real-time quantitative polymerase chain reaction （RTQ-PCR） assay based on SYBR Green I technology was developed to target 3D gene of DHV-1, Comparative sequence analysis showed that the genome has a typical picornarivus genetic organization, and strain DHV-1 R genetic organaiztion is 5＇ untranslated region （UTR）-VP0-VP3-VPI-2A1-2A2-2B-2C-3A-3B-3C-3D-3＇ UTR, DHV-1 R has close relationship with Parechovirus, and has 95.1-99.1% nucleotide sequence identity with other DHV-1 strains. Based on the DHV-1 sequences in GenBank, three pairs of specific primers were designed to amplify DHV-1 using real-time PCR. The results showed that real-time PCR Tm value is 85.6℃ and the real-time PCR provides a broad dynamic range, detecting from 102 to 109 copies of DHV-1 cDNA per reaction. No cross-reactions were found in specimens containing DPV, AIV and NDV. It is concluded that DHV-1 belongs to a new group of the family Picornaviridae that may form a separate genus most closely related to the genus Parechovirus. All results showed that the real-time PCR has high sensitivity and specificity to detect DHV-1 using SYBR Green I dissociation curve analysis, isolates can be distinguished by their melting temperature. These methods are rapid, sensitive, and reliable, and can be readily adapted for detection of DHV-1 from other clinical samples.