Construction of cytopathic PK-15 cell model of classical swine fever virus

No cytopathic effect (CPE) can be observed on classical swine fever virus (CSFV) infected cell culture in vitro. This brings an obstacle to the researches on reciprocity between CSFV and host cells. Based on the construction of full-length genomic infectious cDNA clone of Chinese CSFV standard virulent Shimen strain, partial deletion is introduced into genomic cDNA to obtain a 7.5 kb subgenomic cDNA. A new subgenomic CSFV is derived from transfection with the subgenomic cDNA on PK-15 cells pre-infected by CSFV Shimen virus. Typical CPE induced by this subgenomic virus is observed on PK-15 cells. Coexistence of wildtype and subgenomic virus in cytopathic cell culture is demonstrated by RT-PCR detection in cytopathic cells. For conclusion, the construction of cytopathic cell model exploited a new way for researches on the molecular mechanism of CSFV pathogenesis.     

Candidate Multi-Peptide-Vaccine Against Classical Swine Fever Virus Induces Strong Antibody Response with Predefined Specificity

IntroductionClassical swine fever virus(CSFV) is a pestiviruswhich causes significantmortality and morbidity inpigs.An epidemic of CSFV in a high pig densityarea can result in devastating financial losses,because few infected pigs can be effectively cured.In many countries in Europe and Asia,classicalswine fever (CSF) is controlled by vaccinationwith commercially available vaccine strains,suchas the Chinese vaccine strain(C-strain,i.e.,hogcholera lapinized virus) orsome modified-live vir…     

Three-dimensional reconstruction of mature dengue virus cultured in neutral medium

Dengue virus (DEN), a single positively stranded RNA virus, is a family member of Flaviviridae; it uses the mosquito Aeodes aegypti as its principle vector to cause what is known as Dengue fever. Its genome is -10.8-kb in size and has one open reading frame encoding three structural proteins: capsid (C), precursor of the membrane (prM) and envelope (E), as well as seven non-structural proteins from a single polypeptide. The immature DEN particles are assembled on the inner surface of the endoplasmic reticulum(ER) and budded into the lumen of the ER.     

Genome sequencing and characterization analysis of a Beijing isolate of chicken coronavirus infectious bronchitis virus

Avian infectious bronchitis virus (AIBV) is classified as a member of the genus coronavirus in the family coronaviridae. The enveloped virus has a positive-sense, single-stranded RNA genome of approximately 28 kilo-bases,which has a 5‘ cap structure and 3‘ polyadenylation tract.The complete genome sequence of infectious bronchitis virus (IBV), Beijing isolate, was determined by cloning sequencing and primer walking. The whole genome is 27733 nucleotides in length, has ten open reading frames:5′-orfla-orflab-s-3a-3b-e-m- 6a-6b-n-3′. Alignments of the genome sequence of IBV Beijing isolate with those of two AIBV strains and one SARS coronavirus were performed respectively. The genome sequence of IBV Beijing isolate compared with that of the IBV strain LX4 (uncompleted, 19440 bp in size) was 91.2% similarity. However, the full-length genome sequence of IBV Beijing isolate was 85.2% identity to that of IBV Strain Beaudette, and was only 50.8% homology to that of SARS coronavirus. The results showed that the genome of IBV has remarkable variation. And IBV Beijing isolate is not closely related to SARS coronavirus. Phylogenetic analyses based on the whole genome sequence, S protein, M protein and N protein, also showed that AIBV Bering isolate is lone virus in group Ⅲ and is distant from SARS coronavirus. In conclusion, this study will contribute to the studies of diagnosis and diseases control on IBV in China.     

Coat protein gene and 3′ non-coding region of tobacco mosaic virus and tomato mosaic virus are associated with viral pathogenesis in Nicotiana tabacum

The camellia isolate of tomato mosaic virus (ToMV-TL) can induce local necrotic lesions on the inoculated leaves in Nicotiana tabacum, whereas the broad bean isolate of tobacco mosaic virus (TMV-B) produces the mosaic symptom on systemic leaves. To examine viral determinant for differential infection phenotype in N. tabacum, the coat protein gene and the 3′ non-coding region of TMV was replaced with that of ToMV, the chimeric virus induced similar local necrotic lesions to that induced by ToMV. The results indicate that the coat protein gene and the 3′ non-coding region of TMV and ToMV influence the virus-induced pathogenesis in N. tabacum.     

A complete sequence and comparative analysis of a SARS-associated virus （Isolate BJO1）

The genome sequence of the Severe Acute Respiratory Syndrome (SARS)-assoclated virus provides essential information for the identification of pathogen(s), exploration of etiology and evolution, interpretation of transmission and pathogenesis, development of diagnostics, prevention by future vaccination, and treatment by developing new drugs.We report the complete genome sequence and comparative analysis of an isolate (B J01) of the coronavirus that has been recognized as a pathogen for SARS. The genome is 29725 nt in size and has 11 ORFs (Open Reading Frames). It is composed of a stable region encoding an RNA-dependent RNA polymerase (composed of 20RFs) and a variable region representing 4 CDSs (coding sequences) for viral structural genes (the S, E, M, N proteins) and 5 PUPs (putative uncharacterized proteins). Its gene order is identical to that of other known coronaviruses. The sequence alignment with all known RNA viruses places this virus as a member in the family of Coronaviridae. Thirty putative substitutions have been identified by comparative analysis of the 5 SARS-associated virus genome sequences in GenBank. Fifteen of them lead to possible amino acid changes (non-synonymous mutations) in the proteins. Three amino acid changes, with predicted alteration of physical and chemical features, have been detected in the S protein that is postulated to be involved in the immunoreactions between the virus and its host.Two amino acid changes have been detected in the M protein,which could be related to viral envelope formation. Phylogenetic analysis suggests the possibility of non-human origin of the SARS-associated viruses but provides no evidence that they are man-made. Further efforts should focus on identifying the etiology of the SARS-associated virus and ruling out conclusively the existence of other possible SARS-related pathogen(s).     

Orisin and future distribution of the new A （H 1N1 ） influenza virus emerging in North America in 2009

The origin of the new A （H1N1） influenza virus recently emerging in North America is a hot controversial topic of significance in disease control and risk assessment. Some experts claimed that it was an unusually mongrelized mix of human, avian and swine influenza viruses, while some others concluded that it was totally a simple re-assortment hybrid of two lineages of swine influenza viruses. Here the phylogenetic diversity of the viral PB1, PA and PB2 gene sequences using online web servers, and the results suggest that all the 8 genetic segments of the new virus were possibly from two lineages of swine influenza viruses, and one of the lineage was a mongrelized mix of human, avian and swine influenza viruses emerging in the world approximately 10 years ago. Considering the recent epidemiological trends of the new virus, we believe it will spread more widely in the world and persist long in human populations. It also could spread among swine populations. The future wide spreading of the new virus may coincide the disappearance of a subtype of previous human influenza A virus.     

Genome evolution of novel influenza A （H1N1 ） viruses in humans

The epidemic situation of A H1N1 flu arose in North America in April 2009, which rapidly expanded to three continents of Europe, Asia and Africa, with the risk ranking up to 5. Until May 13th, the flu virus of A H1N1 had spread into 33 countries and regions, with a laboratory confirmed case number of 5728, including 61 deaths. Based on IRV and EpiFluDB database, 425 parts of A H1N1 flu virus sequence were achieved, followed by sequenced comparison and evolution analysis. The results showed that the current predominant A H1N1 flu virus was a kind of triple reassortment A flu virus： （i） HA, NA, MP, NP and NS originated from swine influenza virus; PB2 and PA originated from bird influenza virus; PB1 originated from human influenza virus. （ii） The origin of swine influenza virus could be subdivided as follows： HA, NP and NS originated from classic swine influenza virus of H1N1 subtype; NA and MP originated from bird origin swine influenza virus of H1N1 subtype. （iii） A H1N1 flu virus experienced no significant mutation during the epidemic spread, accompanied with no reassortment of the virus genome. In the paper, the region of the representative strains for sequence analysis （A/California/04/2009 （H1N1） and A/Mexico/4486/2009 （H1N1）） included USA and Mexico and was relatively wide, which suggested that the analysis results were convincing.     

Molecular phylogeny of coronaviruses including human SARS-CoV

Phylogenetic tree of coronaviruses (CoVs) including the human SARS-associated virus is reconstructed from complete genomes by using our newly developed Kstring composition approach. The relation of the human SARS-CoV to other coronaviruses, i.e. the rooting of the tree is suggested by choosing an appropriate outgroup.SARS-CoV makes a separate group closer but still distant from G2 (CoVs in mammalian host). The relation between different isolates of the human SARS virus is inferred by first constructing an ultrametric distance matrix from counting sequence variations in the genomes. The resulting tree is consistent with clinic relations between the SARS-CoV isolates. In addition to a larger variety of coronavirus genomes these results provide phylogenetic knowledge based on independent novel methodology as compared to recent phylogenetic studies on SARS-CoV.     

Value of ecosystem services in China

The function and services are the important components of the life-support system in the planet, as well as the basic elements for sustainable development of environment and society.It is a must to evaluate it for incorporating it with the social-economic system.It is also an important approach to draw the public attention on the environmental and ecosystem conservation.In this study, the ecosystem function and services in China were estimated by employing the classification and economic parameters from Costanza et al.The type and area of terrestrial ecosystems were extracted from Vegetation Map of China (1:4 000 000), and then the distribution map of ecosystem services of China was drawn.According to our calculation, the total value of ecosystem services in China is 77 834.48′108 RMB yuan per annum.The value for terrestrial ecosystem is 56 098.46′108 yuan per annum, and that for marine ecosystem is 21 736.02′108 yuan per annum.The value of ecosystem services in China is 1.73 times bigger than GDP in 1994.The value for forest ecosystem services is 15 433.98′108 yuan per annum, which is 27.51% of the total annual ecosystem services in China.Although wetland is little in area, its ecosystem service value is huge, which is 26 736.9′108 yuan per annum.The value for grassland ecosystem is 8 697.68′108 yuan per annum.Coastal ecosystem service is 12 223.04′108 yuan per annum.Overall, the ecosystem service in China contributes 2.71% to that of our planet.The estimation method employed in this study was a conservative one, and should be improved in the future studies.     

Infective viruses produced from full-length complementary DNA of swine vesicular disease viruses HK/70 strain

Swine vesicular disease (SVD) is a highly conta- gious viral disease of pigs. Symptoms are clinically indistinguishable from those caused by other vesiculardisease viruses, such as foot and mouth disease (FMD virus, vesicular stomatitis (VS) virus and ves…     

猪瘟强毒株与兔化弱毒疫苗株的LAMP鉴别检测

为了鉴别猪瘟病毒（CSFV）野毒感染和疫苗接种,系统比较分析CSFV标准强毒株、疫苗株、不同基因亚型的野毒株的全基因组序列差异,设计一套分别针对猪瘟强毒与弱毒NS5B基因的环介导等温扩增（LAMP）引物,建立特异性的猪瘟强毒株与疫苗株的LAMP检测方法.LAMP扩增产物用罗丹明B指示剂或者琼脂糖凝胶电泳进行检测.该方法特异性好,比RT-PCR灵敏度高出1 000倍,且重复性稳定性良好,为快速准确地鉴别CSFV野毒感染和疫苗接种提供了有效的方法.     

Construction and identification of reverse genetics system of Dengue type 2 virus isolated in China

Dengue (DEN) viruses, mosquito-borne pathogens of the Flavivirus genus, Flaviviridae family, are envel- oped RNA viruses that contain a single-stranded, posi- tive-sense, capped RNA genome of approximately 11 kb. Single polypeptide is co-translationlly pr…     

Construction of cDNA library, nucleotide sequence and analysis of entire genome of classical swine fever virus strain Shimen

According to the previously published CSFV sequences, 18 pairs of primers have been designed and synthesized, which cover the entire genome of CSFV strain Shimen. Each cDNA fragment has been amplified by RT-PCR from the anticoagulant blood of strain Shimen infected pig. The PCR products have been cloned respectively and sequenced. Results show that the cDNA library of strain Shimen and its nucleotide sequence have been obtained. The genomic RNA of strain Shimen is 12 298 nucleotides in length, containing a 5′ and a 3′ noncoding region 373 and 231 nt long respectively. The center of genome is a single large open reading frame of 11 697 nt which encodes a polyprotein of 3 898 amino acids. The entire sequence of strain Shimen has also been compared with that of other CSFV strains.     

Infectious foot-and-mouth disease virus derived from a cloned full-length cDNA of OH／CHA／99

The China foot-and-mouth virus (FMDV) isolate OH/CHA/99 was isolated from swine, which was unable to infect bovine thyroid cells in vitro or to cause typical disease in bovines following intradermal inoculation in the tongue. To enhance antigenicity, replication, maturation and pathogenicity studies of OH/CHA/99, an infectious fulllength cDNA clone, designated pBIFMDV, was prepared. The in vitro and in vivo biological properties of the virus derived from pBIFMDV were studied by analyzing antigenicity, plaque morphology and virulence in pigs. The results showed that the virus derived from pBIFMDV had the same biological properties as the parent strain OH/CHA/99; the fulllength infections cDNA clone, pBIFMDV, will be very useful in studies of the antigenicity, virulence, pathogenesis, maturation and replication of FMDV.     

快速检测猪瘟兔化弱毒疫苗株的TaqMan荧光定量RT-PCR方法的建立

在猪瘟病毒兔化弱毒疫苗株（HCLV）的5′端非编码区设计一对引物和一条TaqMan探针,通过优化反应条件,成功建立了特异性检测HCLV的荧光定量RT-PCR方法．结果表明：该方法检测的最低拷贝数为45拷贝／μL,灵敏度比普通PCR方法高10^4倍,在较广的范围内（4．5×10^1-4．5×10^6拷贝／μL）有良好的线性关系（r=0．994）;分别以乙型脑炎病毒、猪繁殖与呼吸综合征病毒、副猪嗜血杆菌、牛病毒性腹泻,黏膜病病毒作为模板进行TaqMan RT-PCR扩增,未出现阳性信号：4个不同浓度标准品组内试验变异系数为1．90％～5．82％,组间试验变异系数为4．02％～5．69％：HCLV3个cDNA样本组内试验变异系数为3．72％～4．93％;组间试验变异系数为2．99％～4．02％．该方法具有很好的灵敏性、特异性及稳定性,能够快速准确定量检测HCLV,为HCLV疫苗的研制、猪瘟病毒分子生物学等方面研究提供了一种快捷有效的工具．     

乙型脑炎病毒(JEV)免疫荧光(IFA)检测方法的建立

目的建立乙型脑炎病毒(JEV)免疫荧光(IFA)检测方法,应用于猪及猪源性生物材料JEV的检测。方法滴定病毒TCID50,筛选JEV敏感细胞,依据国标IFA法制备抗原片,并进行特异性、敏感性和稳定性试验。结果选取BHK21细胞作为JEV敏感细胞,病毒感染力滴度(TCID50)为10-8.9.mL-1;与猪瘟病毒(CSFV)、猪细小病毒(PPV)均无交叉反应;稳定性和敏感性试验显示,不同时间IFA检测灵敏度均为1∶2560;可检测到的病毒滴度最低为10-6.5.mL-1。结论建立的IFA法敏感性、特异性强,稳定性好,可用于猪及猪源性生物材料JEV的检测。