Genomic characterization and pathogenicity of a porcine hemagglutinating encephalomyelitis virus strain isolated in China

Porcine hemagglutinating encephalomyelitis virus (PHEV) is a member of the genus betacoronavirus within the family coronaviridae, which invades the central nervous system (CNS) via peripheral nervous system and causes encephalomyelitis or vomiting and wasting disease (VWD) in sucking piglets. Up to now, although few complete nucleotide sequences of PHEV have been reported, they are not annotated. This study aimed to illuminate genome characterization, phylogenesis and pathogenicity of the PHEV/2008 strain. The full length of the PHEV/2008 strain genome was 30,684 bp, with a G?+?C content of 37.27%. The genome included at a minimum of 11 predicted open reading frames (ORFs) flanked by 5′ and 3′ untranslated regions (UTR) of 211 and 289 nucleotides. The replicase polyproteins pp1a and pp1ab, which had 4382 and 7094 amino acid residues, respectively, were predicted to be cleaved into 16 subunits by two viral proteinases. Phylogenetic analysis based on the complete genome sequence revealed that PHEV/2008 strain was genetically different from other known PHEV types, which represented a novel genotype (GI-1). In addition, we found that PHEV/2008 was neurotropic and highly pathogenic to 4-week-old BALB/c mice. Taken together, this is the first detailed annotated, complete genomic sequence of a new genotype PHEV strain in China.     

Isolation and genomic characterization of three enterovirus 90 strains in Shandong, China

Enterovirus 90 (EV90) is a newly identified serotype of the species Human enterovirus A, and few nucleotide sequences of EV90 are available. In this study, three EV90 strains were isolated from acute flaccid paralysis (AFP) cases in Shandong Province, China, in 2001 and 2003. Sequence analysis revealed 96.7–98.0 % VP1 nucleotide identity among themselves and 77.7–92.3 % to other EV90 strains. Complete genome analysis provided evidence of recombination in the non-capsid coding region of strain 01421. This is the first report of EV90 in China, and the low isolation rate suggests that it has not been a prevalent serotype in China.     

Complete genome analysis of human enterovirus B73 isolated from an acute flaccid paralysis patient in Shandong,China

Human enterovirus B73 (EV-B73) is a member of species Enterovirus B. To date, only one complete genome sequence of prototype strain CA55-1988 from California has been available. In this study, the complete genome analysis of an EV-B73 strain 088/SD/CHN/04 isolated from an acute flaccid paralysis case in Shandong Province, China in 2004 is conducted. It had 75.6 and 79.3 % nucleotide similarity with prototype strain CA55-1988 in the VP1 coding region and the complete genome, respectively. It had great VP1 nucleotide divergence (16.7–24.4 %) with EV-B73 strains from other parts of the world. Similarity plot and bootscanning analyses provided evidence of recombination with other EV-B viruses.     

无菌性脑膜炎暴发中柯萨奇B3病毒的基因特征分析

目的 明确2008年山东省临沂市郯城县无菌性脑膜炎暴发的病原,对分离到的柯萨奇B3病毒的基因特征、遗传变异规律及进化来源进行分析.方法 采集暴发病例的粪便、脑脊液标本,应用RD、Hep-2细胞进行病毒分离;阳性分离物分别采用中和试验、逆转录-聚合酶链式反应(RT-PCR)和核苷酸序列测定方法进行定型,最后与GenBank中检索到的其他CVB3毒株进行同源性分析,并构建VP1完整编码区亲缘进化树研究其遗传进化规律.结果 共采集22份粪便和120份脑脊液标本,分离到病毒35株,经鉴定34株为CVB3,1株为ECH030.34株CVB3分离株VP1区部分序列(381 bp)核苷酸同源性为90.5%～100%,氨基酸同源性为98.4%～100%;与Nancy原型株的核苷酸同源性为79.5%～81.6%,氨基酸同源性为98.4%～99.2%.012/2008TC/SD/CHN和177/2008TC/SD/CHN与安徽2008年分离的Fuyang19株同源性最高,核苷酸同源性分别为98.2%和91.0%,氨基酸同源性分别为99.2%和98.9%;进化树分析显示,中国大陆分离株独处一支,且呈单源性发生关系.结论 此次脑膜炎暴发的病原是CVB3.它与其他的中国分离株同源性高,与国外分离到的CVB3具有不同的基因特征.     

Molecular characterization of an infectious bronchitis virus strain isolated from northern China in 2012

This study reports the complete genome sequence of an infectious bronchitis virus (CK/CH/SD/121220, KJ128295) isolated in 2012 from Shandong Province in northern China. The genome is 27,666 nt long, comprising six genes and 5′ and 3′ untranslated regions. The full-length genome of the CK/CH/SD/121220 isolate had the highest nucleotide sequence identity (96.7 %) to the YX10 strain. Sites of recombination were identified in the genes 1ab, S, 5a, 5b and N, with their putative parental strains belonging to the QX- and YN-type subgroups, which are already circulating in China. Our findings suggest an important role played by recombination in IBV evolution.     

Genomic and proteinic characterization of strain S, a rickettsia isolated from Rhipicephalus sanguineus ticks in Armenia.

Strain S, a spotted fever group (SFG) rickettsia isolated from Rhipicephalus sanguineus ticks collected in Armenia, was identified. Microimmunofluorescence, sodium dodecyl sulfate-polyacrylamide gel protein electrophoresis and Western immunoblotting, PCR and then restriction fragment length polymorphism analysis, pulsed-field gel electrophoresis, and 16S rRNA gene sequencing were used to compare strain S with reference isolates. Strain S was found to possess proteinic, antigenic, and genomic patterns which were unique among SFG rickettsiae. Strain S is characterized by its high degree of pathogenicity for experimental animals, but its role as a potential human pathogen should be determined. The role of R. sanguineus ticks in the epidemiology of SFG rickettsiae is discussed.     

Genomic analysis of Newcastle disease virus strain NA-1 isolated from geese in China

Newcastle disease virus (NDV) has been thought to infect only domestic avian species, with waterfowl such as geese either not being infected, even by virulent strains, or developing only in apparent infection. In 1997, a new infectious disease producing high morbidity and mortality among geese broke out in many provinces of China, which was caused by a serotype I avian paramyxovirus (APMV-1)--NDV. To investigate how NDV spreads between chickens and geese, the complete genome of one NDV strain isolated from a goose was cloned and analyzed. The results indicate that there is conservation in NDV structural genetic evolution but that there are also considerable differences between goose and chicken NDV strains. Separate patterns of NDV evolution exist among wild bird species. Meanwhile, there is evidence indicating that the goose NDV may have evolved from chicken NDV strain Herts/33. In addition, the possibility was investigated that this new strain of NDV may bind to different sialic acid receptor binding sites than the normal NDV strains that have been investigated so far. This might provide clues to the evolution of the goose NDV.     

Genomic sequence of an avian paramyxovirus type 1 strain isolated from Muscovy duck (Cairina moschata) in China

The complete sequence of an avian paramyxovirus type 1 (APMV-1) strain, FP1/02, isolated from Muscovy duck in China, was determined. Sequence analysis indicated that the complete genome of strain FP1/02 contained 15,192 nucleotides (nt), following the rule of six. The genome contained an extra 6-nt insertion in the non-coding region of the NP gene when compared with other APMV-1 strains, such as strains La Sota and Beaudette C. The cleavage site of the F protein was (112)R-R-Q-K-R↓F(117), indicating that the FP1/02 strain was virulent, but the morbidity and mortality varied with the species of duck. Genotypic analysis based on the F gene revealed that APMV-1 FP1/02 was a member of genotype VII. Phylogenetic analysis showed that the FP1/02 strain shared high identity with other APMV-1 strains such as ZJ1, SF02 and NA-1 isolated from geese.     

Molecular identification of an enterovirus 99 strain in Spain

Enterovirus 99 is a recently described genotype of virus belonging to the species Human enterovirus C. So far, only a few sequences of this enterovirus type have been available. In 2010, during Spanish enterovirus surveillance, an enterovirus 99 strain was found in an acute flaccid paralysis patient. The virus was detected and typed in the clinical samples using molecular methods. Phylogenetic analysis in the 3Dpol region revealed recombination events with other species-C enteroviruses. This is the first finding of this unusual type in Spain.     

Genomic characterization of an endemic Mycobacterium tuberculosis strain: evolutionary and epidemiologic implications

In a study of 302 Mycobacterium tuberculosis clinical isolates from the low-incidence Canadian-born population of Quebec, we characterized a large endemic strain family by using genomic deletions. The DS6(Quebec) deleted region (11.4 kb) defined a strain family of 143 isolates encompassing two subgroups: one characterized by pyrazinamide (PZA) susceptibility and the other marked by a PZA-monoresistant phenotype. A second deletion (8 bp) in the pncA gene was shared by all 76 isolates with the PZA resistance phenotype, whereas a third DRv0961 deletion (970 bp) defined a further subset of 15 isolates. From their deletion profiles, we derived a most parsimonious evolutionary scenario and compared multiple standard genotyping modalities (using IS6110 restriction fragment length polymorphism [RFLP], spoligotyping, and mycobacterial interspersed repetitive units [MIRU]) across the deletion-based subgroups. The use of a single genotyping modality yielded an unexpectedly high proportion of clustered isolates for a high IS6110 copy strain (27% by IS6110 RFLP, 61% by MIRU, and 77% by spoligotyping). By combining all three modalities, only 14% were genotypically clustered overall, a result more congruent with the epidemiologic profile of reactivation tuberculosis, as suggested by the older age (mean age, 60 years), rural setting, and low proportion of epidemiologic links. These results provide insight into the evolution of genotypes in endemic strains and the potential for false clustering in molecular epidemiologic studies.     

肠道病毒71型山东临沂分离株全基因组序列分析

目的 自1例死亡患儿的标本中分离EV71,并分析其全基因组序列特点,研究其基因序列的改变是否与其神经毒力有关.方法 咽拭子标本采自山东临沂市人民医院1例死亡患儿,在人横纹肌瘤细胞(RD)上分离EV71,分段扩增获得EV71的全序列,用BLAST、Bioedit和MEGA 4进行序列分析.结果 获得1株EV71分离株SDLY107,基因组全长7405 bp,全基因组核苷酸序列与2008年的阜阳株Fuyang.Anhui.P.R.C/17.08/2同源性最高,为98.6%,与原型株BrCr/70的同源性为80.0%,与神经毒型株MS/87的同源性为86.5%.系统进化分析表明,SDLY107与中国大陆的北京株、河南株、广西株、深圳株、兰州株、阜阳株、重庆株、浙江株的亲缘关系较近,按照传统的VP1基因分型方法,可归为C4亚型,是近年来我国大陆流行的主要基因亚型.氨基酸序列分析发现,SDLY107与其他毒株相比,有2个特有的突变(E947D,K1873R).结论 SDLY107分离株属于C4亚型,氨基酸突变E947D和K1873R可能与EV71的致病性有关.

Abstract：

Objective To isolate enterovirus 71 from a death children,and analyze whether the neurovirulence was related to the variation of nucleotide and amino acid. Methods Enterovirus 71 was isolated from throat swabs which were colleted from Shandong Linyi People's Hospital. The full length genome was sequenced by amplification with RT-PCR and sequencing of 9 overlapped gene fragments covering full length of the genomes. The nucleotide and amino acid sequenced was aligned by BLAST, Bioedit and MEGA 4. Results A strain of enterovirus 71 was isolated and named as SDLY107. The full length was 7405 bp. The results of homology analysis of overall nucleotide sequence showed that strain Fuyang. Anhui. P. R. C/17.08/2 had highest homology (98.6%)with strain SDLY107, and the homology was 80.0% between strain SDLY107 with prototype strain BrCr/70,and 86. 5% between strain SDLY107 with nerve strain MS/87. Phylogenetic analysis showed that the phylogeny was close between SDLY107 with some isolated strains from Chinese Mainland, such as Beijing, Henan, Guangxi, Sbenzhen, Lanzhou, Fuyang, Chongqing and Zhejiang strains, which was clustered for C4 subtype. The results of amino acid sequence analysis showed that there were 2 mutations, E947D and K1873R, for strain SDLY107. Conclusion SDLY107 belonged to C4 subtype, amino acid mutations E947D and K1873R of which may be relevant to the pathogenicity of EV71.     

Genomic characterisation of a lentogenic Newcastle disease virus strain HX01 isolated from sick pigs in China

This paper describes the complete genome sequence of HX01, an isolate of the Newcastle disease virus (NDV) collected from a swine disease outbreak. The genome is 15,186 nt long and consists of six genes in the order of 3′-NP-P-M-F-HN-L-5′. This genome has the same length as the old NDV genotypes (I–IV), whereas the new NDV genotypes (V–IX) are 15,192 nt long. Compared with the genomic sequences of the reference NDV strains, the HX01 genome is highly similar to the genome of other NDV strains. However, some unique features of the HN gene were found in HX01. HX01 possesses the motif ¹¹²G-R-Q-G-R-L¹¹⁷ at the fusion protein cleavage site, which is typical of lentogenic strains. Pathogenicity tests based on the mean death time and the intracerebral pathogenicity index also revealed the isolate’s lentogenic character. Phylogenetic analysis based on the variable region of the F gene (nt 47–420) revealed that HX01 was clustered to genotype II within class II NDV. Genetically, HX01 has a high similarity with the La Sota vaccine strain based on the single gene or complete genomic but is far different from the prevalent genotype VIId NDV which circulates in fowls and waterfowls in mainland China.     

中国人幽门螺杆菌尿素酶B亚单位的基因克隆及序列分析

目的 克隆人幽门螺杆菌（ｈｅｌｉｃｏｂａｃｔｅｒ ｐｙｌｏｒｉ,Ｈｐ）尿素酶Ｂ基因（ｕｒｅＢ）,并分析其核苷酸序列的特性。方法 用ＰＣＲ技术从临床分离的Ｈｐ菌株基因组中扩增出ｕｒｅＢ基因,将其克隆至ｐＨＰ质粒上进行序列分析。结果 克隆得到的ｕｒｅＢ基因长度为１７１３ｂｐ,其核苷酸序列与ＧｅｎＢａｎｋ公布的序列有６１个碱基存在差异,同源为９６４４％,推定的氨基酸序列同源性为９９．６５％。结论：我们所     

Genomic characterization of a poxvirus isolated from a child

A poxvirus was isolated from a six-year-old girl. The comparative analyses of the genome of this isolate (H-CP-LSax) which were carried out using the restriction endonucleases BamHI, HindIII, KpnI, MluI, NcoI, SacI, and SmaI revealed that this isolate is a member of the genus orthopoxvirus. Since the girl had never been vaccinated against smallpox, and had close contact to domestic animals, including cats, rabbits and guinea pigs, the genome of H-CP-LSax virus was genetically analysed in comparison with other known orthopoxviruses. The analysis demonstrates clearly that the HindIII clevage pattern of H-CP-LSax DNA is different from the HindIII DNA cleavage patterns of vaccinia virus, cowpox virus, rabbit poxvirus, cat poxvirus, ectromelia virus, and okapi poxvirus. Surprisingly, it was found that the HindIII and SmaI cleavage patterns of the DNA of one out of six elephant poxviruses which were analysed under the same conditions were virtually identical to the HindIII and SmaI cleavage patterns of H-CP-LSax DNA. Although SmaI and HindIII digestion of both virus genomes gave the same fragment patterns, the viral DNAs can be distinguished from each other by the restriction endonucleases SacI, BamHI, and KpnI, which also show high similarities in the fragmentation patterns of both viruses. The results obtained in this study indicate three possibilities concerning the origin of H-CP-LSax virus. Firstly that the H-CP-LSax virus originated from an unknown animal species. Secondly, that this virus is a variant of elephant poxvirus in which the HindIII and SmaI sites are extremely conserved, and finally that H-CP-LSax can be a recombinant virus of unknown origin.     

Genomic characterization of a novel group A lamb rotavirus isolated in Zaragoza, Spain

An ovine rotavirus (OVR) strain, 762, was isolated from a 30-day-old lamb affected with severe gastroenteritis, in Zaragoza, Spain, and the VP4, VP7, VP6, NSP4, and NSP5/NSP6 genes were subsequently characterized molecularly. Strain OVR762 was classified as a P[14] rotavirus, as the VP4 and VP8* trypsin-cleavage product of the VP4 protein revealed the highest amino acid (aa) identity (94% and 97%, respectively) with that of the P11[14] human rotavirus (HRV) strain PA169, isolated in Italy. Analysis of the VP7 gene product revealed that OVR762 possessed G8 serotype specificity, a type common in ruminants, with the highest degree of aa identity (95-98%) shared with serotype G8 HRV, bovine rotavirus, and guanaco (Lama guanicoe) rotavirus strains. Moreover, strain OVR762 displayed a bovine-like NSP4 (genotype E2) and NSP5/NSP6 (genotype H3), and a VP6 genotype I2, as well as a long electropherotype pattern. This is the first report of a lamb rotavirus with P[14] and G8 specificities, providing additional evidence for the wide genetic and antigenic diversity of group A rotaviruses.     

肾综合征出血热山东分离株鲁宁-84刘株的分子特征分析

目的 分析肾综合征出血热山东分离株鲁宁—84刘株的分子特征。方法 应用逆转录聚合酶链反应(RT—PCR)扩增鲁宁—84刘株M和S片段全基因，克隆于T载体，纯化后测定序列，应用DNA STAR软件比较分析。结果 鲁宁—84刘株(JNL株)的M片段的全基因序列共3615个核苷酸，编码1135个氨基酸，S片段的全基因序列共1698个核苷酸，编码429个氨基酸，为HTN型毒株。JNL株与HTN型毒株M和S全基因序列差异分别高达20．0％-20．6％和15．5％-16．0％，基于核苷酸序列的种系发生分析结果也可以看到，鲁宁—84刘株不同于国内外其他的分离株，处于独立的分支。结论 山东地区鲁宁—84刘株流行株是与国内外HTN型毒株不同的新的HTN亚型病毒。