An Experimental Study of a Concurrent Primary Infection with Bovine Respiratory Syncytial Virus (BRSV) and Bovine Viral Diarrhoea Virus (BVDV) in Calves

Experimental infections with bovine respiratory syncytial virus (BRSV) and bovine viral diarrhoea virus (BVDV) were performed to study the effect of concurrent BRSV and BVDV infections. Twelve seronegative calves, in 3 groups, were inoculated on a single occasion with pure BRSV (group A), BRSV and noncytopathogenic BVDV (group B) or mock infected (group C). Mild respiratory symptoms were recorded 4 to 5 days post inoculation (dpi) in group A and group B calves. One calf in group A was severely affected and required medical treatment. In group B, fever (40.7-41.4 °C) was prominent 7 to 8 dpi. Only calves in group B were BVDV positive in purified lymphocytes at 2 to 14 dpi and showed increased serum interferon levels, with a peak at 4 dpi, indicating BVDV to be responsible for inducing the rise. BRSV was detected in lung lavage fluids up to 7 dpi for group A calves, compared to 11 dpi for group B and calves in this group also seroconverted later displaying lower BRSV titers. The time lag before an antibody response and the titers recorded in group B, indicated that the duration of BVDV infection in lymphocytes negatively influenced the capacity to mount a BRSV antibody response. kw|Keywords|k]cattle; k]respiratory disease; k]interferon; k]PCR     

牛病毒性腹泻病毒的成熟和释放

试验中用电镜观察了牛病毒性腹泻病毒ＯｒｅｇｏｎＣ２４Ｖ株在感染新生牛睾丸细胞中的形态发生。成熟的病毒颗粒是直径约为５０ｎｍ的球形颗粒,内含直径约为３０ｎｍ的核心。病毒在宿主细胞的胞质内复制,通过糙面内质网膜出芽成熟。病毒可以通过外排或在细胞死亡后含有病毒颗粒的空泡崩溃而释放到胞外。     

牛病毒性腹泻病毒RT-LAMP检测方法的建立

目的:建立牛病毒性腹泻病毒(BVDv)的环介导体外等温扩增(LAMP)快速检测方法。方法:根据BVDv的5'端非编码区序列,在保守区的8个位点设计LAMP特异性引物(2对特异性引物和1对环引物),对反应条件和试剂浓度进行优化,建立恒温(63.5℃)、快速(65min)的检测方法。结果:建立的方法特异性好,检测其他对照病毒均为阴性;灵敏度高,最低可检测到1个拷贝的阳性质粒,可通过观察浑浊度或加入染料后直接判定扩增结果。结论:建立了用于检测BVDv的LAMP方法,该方法简便、快速、特异性好、灵敏度高,适合基层和现场检测。     

牛病毒性腹泻病毒基因组cDNA文库的构建

以牛病毒性腹泻病毒（ＢＶＤＶ）┥ｎＬ株的基因组ＲＮＡ为模板,经逆向转录酶作用,合成第一链ｃＤＮＡ,再以ＲＮａｄｓｅ／Ｈ与ＤＮＡ聚合酶Ｉ联合作用合成ｄｓｃＤＮＡ,并以ｄＣ同聚物尾化。ｐＵＣ８ＤＮＡ在Ｐｓｔ Ｉ酶解后,以ｄＧ同聚物尾化,两者退火构成重组质粒,转化到Ｅ．ｃｏｌｉ ＪＭ１０１受体菌中,另以γ－＾３２Ｐ－ＡＴＰ标记ＢＶＤＶ ＲＮＡ制备探针,通过菌落原位杂交筛选重组子。酶切分配表明重组质粒插入     

Functional Characterization of Bovine Viral Diarrhea Virus Nonstructural Protein 5A by Reverse Genetic Analysis and Live Cell Imaging

Nonstructural protein 5A (NS5A) of bovine viral diarrhea virus (BVDV) is a hydrophilic phosphoprotein with RNA binding activity and a critical component of the viral replicase. In silico analysis suggests that NS5A encompasses three domains interconnected by two low-complexity sequences (LCSs). While domain I harbors two functional determinants, an N-terminal amphipathic helix important for membrane association, and a Zn-binding site essential for RNA replication, the structure and function of the C-terminal half of NS5A are still ill defined. In this study, we introduced a panel of 10 amino acid deletions covering the C-terminal half of NS5A. In the context of a highly efficient monocistronic replicon, deletions in LCS I and the N-terminal part of domain II, as well as in domain III, were tolerated with regard to RNA replication. When introduced into a bicistronic replicon, only deletions in LCS I and the N-terminal part of domain II were tolerated. In the context of the viral full-length genome, these mutations allowed residual virion morphogenesis. Based on these data, a functional monocistronic BVDV replicon coding for an NS5A variant with an insertion of the fluorescent protein mCherry was constructed. Live cell imaging demonstrated that a fraction of NS5A-mCherry localizes to the surface of lipid droplets. Taken together, this study provides novel insights into the functions of BVDV NS5A. Moreover, we established the first pestiviral replicon expressing fluorescent NS5A-mCherry to directly visualize functional viral replication complexes by live cell imaging.     

Comparison of the Prevalence and Incidence of Infection with Bovine Virus Diarrhoea Virus (BVDV) in Denmark and Michigan and Association with Possible Risk Factors

Based on 2 previous surveys on the occurrence of infection with bovine virus diarrhoea virus (BVDV) in Danish and Michigan dairy herds, the prevalence and incidence of the infection were compared. The presence of certain possible risk factors for the occurrence of infection in the 2 areas were summarized and it was investigated if any of these risk factors had significant effect on the presence of animals persistently infected (PI) with BVDV in the dairy herds. Information on the cattle population density in the 2 areas was obtained from statistical yearbooks. Further information for the individual farms on age distribution, housing of animals, herd size, pasturing and purchasing policy was gathered. The prevalence of PI animals was more than 10 times higher in Denmark as compared to Michigan. In herds without PI animals, the annual incidence of seroconversion as calculated from the age specific prevalence of antibody carriers varied in most age groups between 20–25% in Denmark and between 5–10% in Michigan. All investigated risk factors except for herd size were in favour of a lower prevalence of infection in Michigan. The use of having animals on pasture and at the same time having purchased more than 40 animals within recent 31/2–4 years were significantly associated with presence of PI animals in the dairy herds (p = 0.01) when tested by the Mantel-Haenszel χ2. Using mul-tivariable logistic regression, the occurrence of PI animals was found to be significantly related to the study area (Michigan and Denmark) as well as to herd size and purchase intensity.     

Immune Response to other Agents of Calves Persistently Infected with Bovine Virus Diarrhoea Virus (BVDV)

The ability of calves persistently infected (PI) with bovine virus diarrhoea virus BVDV to respond immunologically to defined antigens other than BVDV was studied. Five clinically healthy PI calves were studied together with 5 non-PI calves serving as controls. The humoral immune response was tested by measuring the serum antitoxin titre following immunization against tetanus. The cellular immune response was tested by the ability to develop a positive reaction in a cutaneous tuberculin test performed 1 month after immunization against Johne’s disease (paratuberculosis). Finally, a skin-sensitizing agent, dinitrochlorobenzene (DNCB), was employed to study whether PI calves would react by hypersensitization following skin exposure to DNCB for 7 consecutive days followed by application of DNCB to a new skin area remote from the area that had first been exposed. The response of PI calves to the various types of antigenic stimuli applied was not significantly different from that of the control calves. Thus, PI calves developed a potent antitoxin response after tetanus immunization, they showed a positive reaction to tuberculin skin test after immunization against paratuberculosis, and were skin sentitized with DNCB.     

Infectious Bovine Viral Diarrhea Virus (Strain NADL) RNA from Stable cDNA Clones: a Cellular Insert Determines NS3 Production and Viral Cytopathogenicity

Bovine viral diarrhea virus (BVDV), strain NADL, was originally isolated from an animal with fatal mucosal disease. This isolate is cytopathic in cell culture and produces two forms of NS3-containing proteins: uncleaved NS2-3 and mature NS3. For BVDV NADL, the production of NS3, a characteristic of cytopathic BVDV strains, is believed to be a consequence of an in-frame insertion of a 270-nucleotide cellular mRNA sequence (called cIns) in the NS2 coding region. In this study, we constructed a stable full-length cDNA copy of BVDV NADL in a low-copy-number plasmid vector. As assayed by transfection of MDBK cells, uncapped RNAs transcribed from this template were highly infectious (>10⁵ PFU/μg). The recovered virus was similar in plaque morphology, growth properties, polyprotein processing, and cytopathogenicity to the BVDV NADL parent. Deletion of cIns abolished processing at the NS2/NS3 site and produced a virus that was no longer cytopathic for MDBK cells. This deletion did not affect the efficiency of infectious virus production or viral protein production, but it reduced the level of virus-specific RNA synthesis and accumulation. Thus, cIns not only modulates NS3 production but also upregulates RNA replication relative to an isogenic noncytopathic derivative lacking the insert. These results raise the possibility of a linkage between enhanced BVDV NADL RNA replication and virus-induced cytopathogenicity.     

牛病毒性腹泻病毒E2蛋白单克隆抗体的制备及初步鉴定

为制备牛病毒性腹泻病毒(BVDV)糖蛋白E2单克隆抗体(MAb),利用原核表达并且纯化的重组糖蛋白E2(rE2)免疫BALB/c小鼠,取免疫后小鼠脾细胞与骨髓瘤细胞SP2/0融合.采用以BVDV为检测抗原的间接ELISA筛选阳性细胞克隆,经3次克隆纯化后获得2株稳定分泌抗E2特异性MAb的杂交瘤细胞株,分别命名为4E3与1G11.用4E3与1G11杂交瘤细胞株接种BALB/c小鼠制备腹水,采用rE2及BVDV包被的ELISA测得的效价分别是6.21×106和6.83×105及6.83×105和7.5×104.间接ELISA、Western blot、IFA试验表明两株杂交瘤细胞所分泌的MAb具有良好的反应性和特异性.经抗体亚类鉴定4E3与1G11均为IgM/K.特异性试验表明4E3与1G11这2株MAb均不与牛传染性鼻气管炎病毒、牛副流感病毒3型、牛腺病毒3型反应;其中4E3不与猪瘟病毒反应,而1G11则可与猪瘟病毒发生交叉反应,这种反应特性可试用于BVDV与猪瘟病毒的鉴别诊断.所制备的4E3与1G11 MAb可以用于BVDV抗原的检测,为建立检测BVDV E2蛋白血清抗体的ELISA奠定了基础.     

A Single Point Mutation in Nonstructural Protein NS2 of Bovine Viral Diarrhea Virus Results in Temperature-Sensitive Attenuation of Viral Cytopathogenicity

For Bovine viral diarrhea virus (BVDV), the type species of the genus Pestivirus in the family Flaviviridae, cytopathogenic (cp) and noncytopathogenic (ncp) viruses are distinguished according to their effect on cultured cells. It has been established that cytopathogenicity of BVDV correlates with efficient production of viral nonstructural protein NS3 and with enhanced viral RNA synthesis. Here, we describe generation and characterization of a temperature-sensitive (ts) mutant of cp BVDV strain CP7, termed TS2.7. Infection of bovine cells with TS2.7 and the parent CP7 at 33°C resulted in efficient viral replication and a cytopathic effect. In contrast, the ability of TS2.7 to cause cytopathogenicity at 39.5°C was drastically reduced despite production of high titers of infectious virus. Further experiments, including nucleotide sequencing of the TS2.7 genome and reverse genetics, showed that a Y1338H substitution at residue 193 of NS2 resulted in the temperature-dependent attenuation of cytopathogenicity despite high levels of infectious virus production. Interestingly, TS2.7 and the reconstructed mutant CP7-Y1338H produced NS3 in addition to NS2-3 throughout infection. Compared to the parent CP7, NS2-3 processing was slightly decreased at both temperatures. Quantification of viral RNAs that were accumulated at 10 h postinfection demonstrated that attenuation of the cytopathogenicity of the ts mutants at 39.5°C correlated with reduced amounts of viral RNA, while the efficiency of viral RNA synthesis at 33°C was not affected. Taken together, the results of this study show that a mutation in BVDV NS2 attenuates viral RNA replication and suppresses viral cytopathogenicity at high temperature without altering NS3 expression and infectious virus production in a temperature-dependent manner.The pestiviruses Bovine viral diarrhea virus-1 (BVDV-1), BVDV-2, Classical swine fever virus (CSFV), and Border disease virus (BDV) are causative agents of economically important livestock diseases. Together with the genera Flavivirus, including several important human pathogens like Dengue fever virus, West Nile virus, Yellow fever virus, and Tick-borne encephalitis virus, and Hepacivirus (human Hepatitis C virus [HCV]), the genus Pestivirus constitutes the family Flaviviridae (8, 20). All members of this family are enveloped viruses with a single-stranded positive-sense RNA genome encompassing one large open reading frame (ORF) flanked by 5′ and 3′ nontranslated regions (NTR) (see references 8 and 28 for reviews). The ORF encodes a polyprotein which is co- and posttranslationally processed into the mature viral proteins by viral and cellular proteases. For BVDV, the RNA genome is about 12.3 kb in length and encodes a polyprotein of about 3,900 amino acids. The first third of the ORF encodes a nonstructural (NS) autoprotease and four structural proteins, while the remaining part of the genome encodes NS proteins which share many common characteristics and functions with the corresponding NS proteins encoded by the HCV genome (8, 28). NS2 of BVDV represents a cysteine autoprotease which is distantly related to the HCV NS2-3 protease (26). NS3, NS4A, NS4B, NS5A, and NS5B are essential components of the pestivirus replicase (7, 10, 49). NS3 possesses multiple enzymatic activities, namely serine protease (48, 52, 53), NTPase (46), and helicase activity (51). NS4A acts as an essential cofactor for the NS3 proteinase. NS5B represents the RNA-dependent RNA polymerase (RdRp) (22, 56). The functions of NS4B and NS5A remain to be determined. NS5A has been shown to be a phosphorylated protein that is associated with cellular serine/threonine kinases (44).According to their effects in tissue culture, two biotypes of pestiviruses are distinguished: cytopathogenic (cp) and noncytopathogenic (ncp) viruses (17, 27). The occurrence of cp BVDV in cattle persistently infected with ncp BVDV is directly linked to the induction of lethal mucosal disease in cattle (12, 13). Previous studies have shown that cp BVDV strains evolved from ncp BVDV strains by different kinds of mutations. These include RNA recombination with various cellular mRNAs, resulting in insertions of cellular protein-coding sequences into the viral genome, as well as insertions, duplications, and deletions of viral sequences, and point mutations (1, 2, 9, 24, 33, 36, 37, 42). A common consequence of all these genetic changes in cp BVDV genomes is the efficient production of NS3 at early and late phases of infection. In contrast, NS3 cannot be detected in cells at late time points after infection with ncp BVDV. An additional major difference is that the cp viruses produce amounts of viral RNA significantly larger than those of their ncp counterparts (7, 32, 50). While there is clear evidence that cell death induced by cp BVDV is mediated by apoptosis, the molecular mechanisms involved in pestiviral cytopathogenicity are poorly understood. In particular, the role of NS3 in triggering apoptosis remains unclear. It has been hypothesized that the NS3 serine proteinase might be involved in activation of the apoptotic proteolytic cascade (21, 55). Furthermore, it has been suggested that the NS3-mediated, enhanced viral RNA synthesis of cp BVDV and subsequently larger amounts of viral double-stranded RNAs may play a crucial role in triggering apoptosis (31, 54).In this study, we describe generation and characterization of a temperature-sensitive (ts) cp BVDV mutant whose ability to cause viral cytopathogenicity at high temperature is strongly attenuated. Our results demonstrate that a single amino acid substitution in NS2 attenuates BVDV cytopathogenicity at high temperature without affecting production of infectious viruses and expression of NS3 in a temperature-dependent manner.     

Microtiter Tests for Detecting Antibody in Bovine Serum to Parainfluenza 3 Virus, Infectious Bovine Rhinotracheitis Virus, and Bovine Virus Diarrhea Virus

Microneutralization tests for detection of antibody in bovine serum to three bovine viruses are described. The Madin-Darby bovine kidney cell line was used with parainfluenza 3 virus (PI 3), whereas serially cultivated bovine embryonic kidney cells were used for infectious bovine rhinotracheitis virus and bovine virus diarrhea virus. Comparison of micro-hemagglutination-inhibition (HI) with micro-serum-neutralization (SN) tests for PI 3 showed the SN test to be more sensitive, more specific, and therefore more useful than the HI test for detecting antibody. Although the effect of trypsin-periodate treatment of serum was to reduce the HI titer of numerous sera by a twofold dilution, sufficient evidence could not be found to indicate that nonspecific HI inhibitors to PI 3 are present in bovine sera.     

Studies on the Decline of Bovine Virus Diarrhoea Virus (BVD V) Maternal Antibodies and Detectability of BVDV in Persistently Infected Calves

Persistently infected (PI) animals play a significant role in spread and transmission of bovine virus diarrhoea virus (B VD V) (Duffell & Harkness 1985). The identification and removal of PI cattle from the herd is of great importance in the control of BVDV. Although PI animals often show various degrees of growth retardation and unthrifty appearance, a significant proportion is clinically normal. PI animals are often seronegative (Duffell & Harkness 1985), but calves may be tested seropositive because of the presence of maternal immunity (Meyling & Jensen 1988). The passively derived BVDV antibodies may interfere with the ability to detect virus. Considering the importance of early recognition of PI calves, it is essential to determine the earliest time when PI animals can safely be diagnosed in the herd.     

牛病毒性腹泻病毒E^rns-E2融合蛋白在果蝇S2细胞中的表达与鉴定

目的：利用果蝇S2细胞表达牛病毒性腹泻病毒（BVDV）Erns-E2融合蛋白,并对其抗体结合能力进行鉴定。方法：用RT-PCR方法扩增BVDV NADL株Erns和E2蛋白的编码基因,利用（G4-S）3柔性15肽基因将扩增的2个基因连接,再与昆虫表达载体pMT/BiP/V5-His连接构建重组表达载体pMT/BiP/V5-His-Erns-E2,将后者与筛选质粒pCoBlast共转染果蝇S2细胞后表达Erns-E2融合蛋白,并对表达产物进行鉴定。结果：SDS-PAGE结果表明,融合蛋白相对分子质量为76800;Western blotting检测表明,该融合蛋白具有与BVDV抗体良好的结合能力。结论：BVDV的Erns-E2融合蛋白能在果蝇S2细胞中进行表达;经鉴定,表达产物具有良好的抗体结合能力,可用于抗原检测。     

牛病毒性腹泻病毒Erns-E2融合蛋白在果蝇S2细胞中的表达与鉴定

目的:利用果蝇S2细胞表达牛病毒性腹泻病毒(BVDV)Erns-E2融合蛋白,并对其抗体结合能力进行鉴定.方法:用RT-PCR方法扩增BVDV NADL株Erns和E2蛋白的编码基因,利用(G4-S)3柔性15肽基因将扩增的2个基因连接,再与昆虫表达载体pMT/BiP/V5-His连接构建重组表达载体pMT/BiP/V5-His-E(MS)-E2,将后者与筛选质粒pCoBlast共转染果蝇S2细胞后表达Erns-E2融合蛋白.并对表达产物进行鉴定.结果:SDS-PAGE结果表明,融合蛋白相对分子质量为76 800;Westem blotting检测表明,该融合蛋白具有与BVDV抗体良好的结合能力.结论:BVDV的Erns-E2融合蛋白能在果蝇S2细胞中进行表达;经鉴定,表达产物具有良好的抗体结合能力,可用于抗原检测.     

Alpha/Beta and Gamma Interferons Are Induced by Infection with Noncytopathic Bovine Viral Diarrhea Virus In Vivo

In contrast to the results of previous in vitro studies, experimental infection of calves with noncytopathic bovine viral diarrhea virus (ncpBVDV) was found to induce strong alpha/beta and gamma interferon responses in gnotobiotic animals. These responses were associated with depressed levels of transforming growth factor beta (TGF-beta) in serum. The results of this study indicate that the immunosuppression caused by ncpBVDV is not associated with low interferon responses or elevated levels of TGF-beta.     

Assignment of the Multifunctional NS3 Protein of Bovine Viral Diarrhea Virus during RNA Replication: an In Vivo and In Vitro Study

Studies on the replication of the pestivirus bovine viral diarrhea virus (BVDV) were considerably facilitated by the recent discovery of an autonomous subgenomic BVDV RNA replicon (DI9c). DI9c comprises mainly the untranslated regions of the viral genome and the coding region of the nonstructural proteins NS3, NS4A, NS4B, NS5A, and NS5B. To assess the significance of the NS3-associated nucleoside triphosphatase/helicase activity during RNA replication and to explore other functional features of NS3, we generated a repertoire of DI9c derivatives bearing in-frame mutations in different parts of the NS3 coding unit. Most alterations resulted in deficient replicons, several of which encoded an NS3 protein with an inhibited protease function. Three lesions permitted replication, though at a lower level than that of the wild-type RNA, i.e., replacement of the third position of the DEYH helicase motif II by either T or F and an insertion of four amino acid residues in the C-terminal part of NS3. While polyprotein proteolysis was found to be almost unaffected in these latter replicons, in vitro studies with the purified mutant NS3 proteins revealed a significantly impaired helicase activity for the motif II substitutions. NS3 with a DEFH motif, moreover, showed a significantly lower ATPase activity. In contrast, the C-terminal insertion had no negative impact on the ATPase/RNA helicase activity of NS3. All three mutations affected the synthesis of both replication products-negative-strand intermediate and progeny positive-strand RNA-in a symmetric manner. Unexpectedly, various attempts to rescue or enhance the replication capability of nonfunctional or less functional DI9c NS3 derivatives, respectively, by providing intact NS3 in trans failed. Our experimental data thus demonstrate that the diverse enzymatic activities of the NS3 protein-in particular the ATPase/RNA helicase-play a pivotal role even during early steps of the viral replication pathway. They may further indicate the C-terminal part of NS3 to be an important functional determinant of the RNA replication process.     

Failure to Induce Mucosal Disease in Cattle Persistently Infected with Bovine Viral Diarrhea Virus by Treatment with Adrenocorticotropic Hormone

Recent research has shown that cattle that develop mucosal disease (MD) often, if not always, have been persistently infected with bovine viral diarrhea virus (BVDV) since birth. The purpose of the present study was to determine whether MD could be induced by immunosuppression of persistently BVDV-infected cattle. For that purpose, adrenocorticotropic hormone (ACTH) was injected intramuscularly, twice daily for 5 consecutive days in 4 persistently BVDV-infected cattle and in 3 control cattle. Before the ACTH treatment, the numbers of leukocytes, neutrophils and mononuclear cells (MNC) per litre of blood in BVDV-infected cattle were in the same range as in the controls. Similarly, the proportions of B cells, T cells, monocytes and Fcγ+ cells (cells with receptor for the Fc part of IgG) were the same in the 2 groups of animals. On the other hand, the proliferative response to mitogen stimulation of MNC obtained from the control animals was twice as high as the corresponding value of the persistently BVDV-infected cattle. In all animals, ACTH treatment caused increased Cortisol concentrations, leukocytosis, neutrophilia and decreased mitogen-induced lymphocyte stimulation. However, the MNC count and the proportions of B cells, T cells, Fcγ+ cells and monocytes remained unaltered. In spite of the immunosuppression, indicated by the decrease in mitogen-induced lymphocyte stimulation. ACTH treatment did not provoke any clinical signs of MD in the persistently BVDV-infected cattle.     

Inhibition of Sphingosine Kinase by Bovine Viral Diarrhea Virus NS3 Is Crucial for Efficient Viral Replication and Cytopathogenesis

Sphingosine 1-phosphate (S1P) is a bioactive sphingolipid implicated in diverse cellular functions including survival, proliferation, tumorigenesis, inflammation, and immunity. Sphingosine kinase (SphK) contributes to these functions by converting sphingosine to S1P. We report here that the nonstructural protein NS3 from bovine viral diarrhea virus (BVDV), a close relative of hepatitis C virus (HCV), binds to and inhibits the catalytic activity of SphK1 independently of its serine protease activity, whereas HCV NS3 does not affect SphK1 activity. Uncleaved NS2-3 from BVDV was also found to interact with and inhibit SphK1. We suspect that inhibition of SphK1 activity by BVDV NS3 and NS2-3 may benefit viral replication, because SphK1 inhibition by small interfering RNA, chemical inhibitor, or overexpression of catalytically inactive SphK1 results in enhanced viral replication, although the mechanisms by which SphK1 inhibition leads to enhanced viral replication remain unknown. A role of SphK1 inhibition in viral cytopathogenesis is also suggested as overexpression of SphK1 significantly attenuates the induction of apoptosis in cells infected with cytopathogenic BVDV. These findings suggest that SphK is targeted by this virus to regulate its catalytic activity.Bovine viral diarrhea virus (BVDV)² is an enveloped, positive-sense single-stranded RNA virus classified in the genus Pestivirus of the family Flaviviridae. BVDV establishes persistent infections in cattle populations worldwide. Because BVDV shares virological and molecular properties with the Flaviviridae family member hepatitis C virus (HCV), which chronically infects an estimated 200 million patients worldwide (1), BVDV is regarded as a surrogate model for HCV (2). Both HCV and BVDV encode a single large precursor polyprotein that is processed by cellular and viral proteases into mature structural and nonstructural (NS) proteins.BVDV NS3 exhibits serine protease and helicase/ATPase activities that require its cofactor NS4A (3). NS3/4A protease is essential for generating mature NS proteins that are required for viral replication. HCV NS3/4A is well characterized and has been shown to suppress type-I interferons by cleaving the cellular interferon mediators IPS-1 and TRIF (4, 5). However, neither interferon suppression nor cellular targets have been identified for the BVDV NS3/4A protease (6).Lytic and persistent BVDV infections depend on the virus biotype. Cytopathogenic (CP) BVDV causes cytopathic effects via apoptosis, whereas noncytopathogenic (NCP) BVDV does not induce obvious changes in cell morphology and viability. These features are distinguished by NS2-3 processing differences; free NS3 produced by NS2-3 cleavage is generated continuously following CP BVDV infections, whereas NS3 is detected only until ∼9 h postinfection (p.i.) for NCP BVDV due to down-regulation of NS2-3 cleavage by this biotype (7). The CP biotype is characterized by dramatic up-regulation of viral RNA synthesis that could be correlated with the induction of cytopathic effect (7–9). Because free NS3, but not NS2-3, can form an active viral replicase complex with other NS proteins, increased viral RNA synthesis promoted through the release of free NS3 has been suggested to be a determinant of the characteristic lytic phenotype of CP BVDV infections (10). However, little is known about the regulation of cellular signaling by BVDV NS2-3, NS3, and NS3/4A, which is crucial for the control of both viral replication and biotype.Recent studies on the mechanisms of viral replication revealed that HCV RNA synthesis occurs on a lipid raft membrane structure where the active viral replicase complex is found (11, 12). The significance of the lipid raft as a scaffold for viral replication is further demonstrated by the identification of a novel HCV replication inhibitor, NA255, which prevents the biosynthesis of sphingolipids, the major components of lipid rafts (13). Administration of NA255 results in disruption of the HCV replicase complexes from the lipid rafts. This report proposes that the interaction between HCV NS5B and sphingomyelin on lipid rafts plays a crucial role for HCV RNA replication. Cellular sphingolipid metabolism is regulated by a large number of converting enzymes that maintain a homeostasis (14) but viral mechanisms that affect the sphingolipid metabolism to facilitate viral replication have yet to be identified.In a search for potential host proteins that interact with BVDV NS3, we identified sphingosine kinase 1 (SphK1) as a binding partner of NS3 using the yeast two-hybrid system. SphK1 is a lipid kinase that catalyzes the phosphorylation of sphingosine to form sphingosine 1-phosphate (S1P), a bioactive sphingolipid implicated in diverse cellular functions, including proliferation, survival, tumorigenesis, development, inflammation, and immunity (14, 15). Here, we analyze the biological significance of the SphK1 interaction with NS3, NS2-3, and NS3/4A. Using purified recombinant SphK1 and NS3, SphK activity was inhibited by NS3 in a dose-dependent manner, independently of its serine protease activity. The inhibition appears to be specific for BVDV NS3 because HCV NS3 had no effect on SphK activity. Using specific chemical inhibitors, small interfering RNA (siRNA), and a catalytically inactive mutant of SphK1, we investigated the significance of SphK inhibition in the viral replication. The present study is the first report demonstrating that SphK1 is targeted by a virus to inhibit its catalytic activity, and this mechanism may contribute to the efficient replication and pathogenesis of BVDV.     

中国4种蝗虫不同种群的遗传分化

采用水平淀粉凝胶电泳技术,应用等位酶分析方法对采自我国山西、江苏、河北等地不同蝗区优势蝗虫种类3科4种8个种群的12种酶18个酶位点进行了检测,比较了4种蝗虫种群水平的等位基因频率变化和它们之间的遗传距离。等位基因频率分析表明：中华稻蝗和笨蝗各位点等位基因丰富,而长翅素木蝗和短额负蝗各位点等位基因较少。在所研究的4种蝗虫8个种群中,多态位点百分率普遍较高(P＝53．3％—100．0％),由于杂合子数目较少而使每个位点的平均杂合度观察值偏低(H。＝0．034—0．139)。受迁飞能力、繁殖方式和活动范围的限制,4种蝗虫的平均杂合度观察值表现出一定的差异：笨蝗较高(H。＝0．089—0．139),其次是中华稻蝗(H。＝0．073—0．090),而长翅素木蝗(H。＝0．0488—0．068)和短额负蝗(H。＝0．034—0．050)相对较低。除Adk-1、Ao-1、Idh-1、Ldh-1、Ldh-2和Me-1在部分蝗虫种群符合Hardy-Weinberg平衡外,其余绝大多数位点的基因型频率显著偏离H—W平衡,但短额负蝗的多个位点符合或接近H—W平衡,表明该种蝗虫在自然种群结构方面明显有别于其他种类。从4种蝗虫的F—统计量(Fst)看,笨蝗种群间基因分化程度最高(Fst＝0．32),其次是短额负蝗(Fst＝0．31),而中华稻蝗相对较低(Fsl＝0．20),长翅素木蝗种群间基因分化程度最低(Fsl＝0．18),利用非加权算术平均法(UPGMA)对I值和D值进行聚类,所得聚类树也证实了这种遗传分化差异,上述结果反映了迁飞能力、适应性和环境因子对不同蝗虫遗传分化的影响。Nei的遗传距离(D)和遗传一致度(I)进行的聚类分析基本符合传统形态学、细胞学等研究结果：即同属于斑腿蝗科的中华稻蝗和长翅素木蝗遗传距离最小(D＝0．559),遗传一致度最高(I＝0．576)。在3个科中,癞蝗科和锥头蝗科之间呈现较小的遗传距离(D＝0．776)和较高的遗传一致度(I＝0．776),而这两科与斑腿蝗科之间的遗传距离相对较大(D＝0．908),遗传一致度相对较低(I＝0．406)。等位酶分析能较好地反映蝗虫不同物种的亲缘关系和种内不同种群之间的遗传分化程度。     

双菱藻科（硅藻门）——四个中国新纪录种

本文报道了采自新疆及云南地区的双菱藻科（硅藻门）中国新纪录植物4种,分别为耳蜗波缘藻Cymato—pleura cochlea Brun、维苏双菱藻Surirella visurgis Hustedt、盾状马鞍藻Campylodiscus clypeus Ehrenberg和莱温德马鞍藻Campylodiscus levanderi Hustedt。通过光镜（LM）和扫描电镜（SEM）观察,对其分类学特征进行了详细的描述,并介绍了他们的生境特征等。