Molecular characterization of RNA and protein synthesis during a one-step growth curve of bovine viral diarrhoea virus in ovine (SFT-R) cells

The aim of this study was to determine the kinetics of noncytopathic bovine viral diarrhoea virus (BVDV) multiplication and synthesis of BVDV specific RNA and proteins in ovine cells (SFT-R) during a one-step growth curve. The virus titre and RNA level were determined by focus-forming assay and real time RT-PCR. The RNA synthesis was detected by Northern blot while synthesis of E2 and NS3 proteins was assayed by immunohistochemistry and Western blot. The results showed that synthesis of viral RNA is initiated at 4 h, NS3 and E2 proteins are detectable at 6-7 h and the replication cycle is complete at 10-12 h. Additionally, we provide evidence that NS2-3 protein was cleaved in ovine cells early during infection and in proliferated leukocytes of acutely infected sheep. This study showed that synthesis of BVDV RNA and proteins in ovine cells occurs at similar times as found in bovine cells.     

Suppression of bovine viral diarrhea virus replication by small interfering RNA and short hairpin RNA-mediated RNA interference

Bovine viral diarrhea virus (BVDV) is a ubiquitous viral pathogen that affects cattle herds' worldwide causing significant economic loss. The current strategies to control BVDV infection include vaccination (modified-live or killed) and control of virus spread by enhanced biosecurity management, however, the disease remains prevalent. With the discovery of the sequence-specific method of gene silencing known as RNA interference (RNAi), a new era in antiviral therapies has begun. Here we report the efficient inhibition of BVDV replication by small interfering (siRNA) and short hairpin RNA (shRNA)-mediated gene silencing. siRNAs were generated to target the 5' non-translated (NTR) region and the regions encoding the C, NS4B and NS5A proteins of the BVDV genome. The siRNAs were first validated using an EGFP/BVDV reporter system and were then shown to suppress BVDV-induced cytopathic effects and viral titers in cell culture with surprisingly different activities compared to the reporter system. Efficient viral suppression was then achieved by bovine 7SK-expressed BVDV-specific shRNAs. Overall, our results demonstrated the use of siRNA and shRNA-mediated gene silencing to achieve efficient inhibition of the replication of this virus in cell culture.     

A Universal `One-tube' RT-PCR Protocol for Amplifying Isolates of Bovine Viral Diarrhoea Virus

The increase in the knowledge of the genetic variability of BVDV and the identification of some of the genetic determinants of its pathogenicity require robust and practical tools for rapid molecular characterization of the various genotypes of this virus. This study was undertaken to develop a standard protocol for RT-PCR that allows the amplification of various parts of the genome of BVDV without the need for optimizing each individual reaction. The reaction set-up is very flexible because it consists of two pre-mixes. These are a master mix, with all the required reagents except the desired primers, which are the components of the second pre-mix and are therefore easily interchangeable between the different reactions. After adding any primer-containing pre-mix to the fixed master mix, a non-interrupted cycling protocol led to the generation of amplicons of up to 4 kbp in size in amounts sufficient for subsequent sequencing reactions. The method was applied to five different regions of the BVDV genome: (i) the well-known 5-UTR to differentiate genotypes I and II; (ii) the entire E2 gene, or an approximately 550 bp region within the E2 gene, in order to find the molecular equivalent of antigenic varieties; (iii) the entire structural protein coding region covering the Npro, capsid, E ^RNS, E1 and E2 genes; (iv) a 2.1 kbp region embracing the NS2/3 junction which is known to be cleaved in cytopathic biotypes of BVDV; and (v) the region covering the entire NS4B and NS5A/B genes. All six RT-PCRs were successfully applied using (i) primers with lengths of between 20 and 52 nucleotides, (ii) an aliquot of RNA extracted from either 10⁶ infected bovine embryonal lung cells or the same number of leukocytes from viraemic cattle, and (iii) all the genotype I and II strains of BVDV tested. The technique described was used to generate various Sindbis virus/BVDV recombinants. The correct processing of the amplicon-derived E2 glycoprotein of BVDV strain PT810 was demonstrated by its reaction with a monoclonal antibody in an immunofluorescence assay. Given the variety of RT-PCRs tested, we conclude that this universal protocol may be useful with other RNA viruses.     

The bovine viral diarrhea virus (BVDV) NS3 protein, when expressed alone in mammalian cells, induces apoptosis which correlates with caspase-8 and caspase-9 activation

The bovine viral diarrhea virus (BVDV) strains exist as two biotypes, cytopathic (cp) and noncytopathic (ncp), according to their effects on tissue culture cells. It has been previously reported that cell death associated to cp BVDV in vitro is mediated by apoptosis. Here, experiments were conducted to determine the involvement of the NS3 protein in the induction of apoptosis. The NS3- and NS3Delta50 (deleted from the NH2-terminal 50 amino acids)-cDNA encoding sequences of BVDV NADL cp reference strain were cloned into adenoviral vectors (AdV) from which the BVDV gene of interest could be expressed from a tetracycline-responsive promoter. A549tTA cells infected in vitro with NS3 or NS3Delta50-expressing AdV showed cytopathic changes characterized by cell rounding and detachment, and nucleus chromatin condensation. DNA fragmentation assays, cytochrome c release, and activation of cellular caspases performed on these infected cells clearly correlated with the observed cytopathic changes with apoptosis. The BVDV NS3Delta50-induced apoptotic process was inhibited by caspase-8- and -9-specific peptide inhibitors (Z-IETD-FMK and Z-LEHD-FMK). Furthermore, apoptosis was inhibited in cells expressing the R1 subunit of herpes simplex virus type 2 ribonucleotide reductase (HSV2-R1) or hsp70, two proteins which are known to inhibit apoptosis associated with caspase-8 activation and cytochrome c release-dependent caspase-9 activation, respectively. Given that HSV2-R1, a specific inhibitor of the caspase-8 activation pathway, efficiently suppressed apoptosis and also prevented caspase-9 activation, the overall results indicate that the BVDV NS3/NS3Delta50 induces apoptosis initiated by caspase-8 activation and subsequent cytochrome c release-dependent caspase-9 activation.     

Cellular insertions in the NS2-3 genome region of cytopathic bovine viral diarrhoea virus (BVDV) isolates

When compared to noncytopathic (ncp) bovine viral diarrhoea virus (BVDV), some cytopathic (cp) BVDV contain additional sequences in the NS2-3 genomic region. One of these insertions, which is 270 nucleotides long and of host origin (cINS), was first described for strain NADL. To find out how frequently this type of insertion occurs in other cp BVDV, 32 cp BVDV field isolates and the BVDV reference cp strain Indiana were screened using RT-PCR which detected cINS in NADL. For most cp viruses an RT-PCR product of 402bp indicated the presence of NS2-3 genes without insertions. In addition, one or two DNA fragments, around 600-850bp in size, were amplified from the genomes of 13 cp viruses indicating the presence of insertions. Sequencing of the PCR products, i.e. 402bp DNA fragment (with no insertion) and longer fragments (with insertion) revealed the location of the insertions in the NS2-3 coding region of eight cp BVDV genomes. All of the insertions were confirmed to be of the cINS type and were located in a very similar position to that found previously in the NADL genome. They were in the same reading frame as the viral polypeptide and they encoded 90-140 amino acids. The 5' and 3' ends of the insertions were different in most of the cp isolates studied. Interestingly, a 14-amino-acid stretch at the 5'-end of the insertion in the cp 5569 isolate as well as 15 amino acids at the 3'-end of the insertion in the cp 5.19516 isolate were not homologous to the cINS sequence. No significant matches for these stretches were found in the EMBL and Swissprot databases.     

牛病毒性腹泻病毒JN株的分离鉴定及E2基因的序列分析

本研究从疑似牛病毒性腹泻病毒(bovine viral diarrhea virus,BVDV)感染牛的分泌物与排泄物中分离鉴定1株牛病毒性腹泻病毒,并进行E2基因序列分析。结果表明,分离株病毒命名为JN株;Reed-Muench法测定分离株病毒TCID₅₀为10^-7.5/0.1 mL;病毒中和试验结果表明,BVDV JN分离株可被BVDV阳性血清特异性中和,而不能被BVDV阴性血清中和;分离株病毒E2基因序列测序结果表明,该分离毒株属于BVDVⅠa亚型。     

牛病毒性腹泻病毒非结构蛋白NS3的表达及ELISA检测方法的建立

本研究旨在建立一种检测牛病毒性腹泻病毒（BVDV）抗体的间接ELISA方法。将BVDV的非结构蛋白NS3基因克隆到原核表达载体pET-32a中进行表达,将纯化后的蛋白作为包被抗原,优化ELISA条件,建立了BVDV抗体间接NS3-ELISA检测方法,并对该方法的特异性、敏感性和重复性进行检测,结果均较好。用所建立的NS3-ELISA方法检测从广西各牛场采集的475份牛血清样品,检出率为24.8%,与商品化试剂盒比较,符合率为97%。结果表明,本研究建立的NS3-ELISA方法简便、快捷,可大批量检测,适用于BVDV的诊断、抗体水平监测及流行病学调查。     

牛病毒性腹泻病毒NS3蛋白纳米抗体的筛选及反应原性检测

本研究旨在获得高效特异性的牛病毒性腹泻病毒（BVDV）NS3（P80）非结构蛋白的纳米抗体。用BVDV灭活疫苗免疫羊驼,测得抗体效价后分离全血中的淋巴细胞。通过噬菌体展示技术构建羊驼重链抗体可变区噬菌体展示文库。经过连续3次吸附-洗脱-扩增的生物筛淘,从中挑选出与BVDV-NS3蛋白结合的噬菌体。对经菌液PCR、琼脂糖凝胶电泳鉴定到的单域抗体（VHH）克隆进行基因测序和同源性比对。用ELISA方法验证筛选出的纳米抗体的反应原性,找到与BVDV-NS3蛋白亲和力高的纳米抗体。结果表明,获得插入率为92.8%、库容为1.84×10¹⁴ CFU/mL的噬菌体展示文库。ELISA结果和氨基酸序列分析显示,成功得到1条与BVDV-NS3蛋白具有良好反应性且与VHH同源性较高的纳米抗体序列。本研究利用大肠杆菌成功表达BVDV-NS3抗原蛋白,建立BVDV纳米抗体噬菌体展示文库,筛选到针对BVDV重要抗原蛋白相应的纳米抗体且与VHH同源性较高。试验结果为牛病毒性腹泻/黏膜病的防控、诊断、治疗及纳米抗体药物的研制提供参考。     

Bovine viral diarrhoea virus: its effects on ovarian function in the cow

Bovine viral diarrhoea virus (BVDV) is a major cattle pathogen responsible for a spectrum of symptoms, including reproductive failure. In this paper we investigate how BVDV interacts with the ovary. The viruses' tropism for the pre-ovulatory oocyte was studied by indirect immunohistochemistry. Two monoclonal antibodies, raised against the non-structural protein NS3 and the envelope glycoprotein E2 were used to probe cryo-sections cut from the ovaries of three persistently infected heifers. NS3 and E2 antigens were widely distributed within the ovarian stroma and follicular cells. NS3 was also localised within the proportion of oocytes. Overall 18.7% of the oocyte population had detectable levels of NS3. What is more, the proportion of antigen positive oocytes remained constant (P>0. 05) throughout the different stages of oocyte maturation.In a subsequent study seven cows were challenged with non-cytopathogenic BVDV (strain Pe515: 5x10(6) TCID(50)) to determine the oestradiol and progesterone responses to an acute infection. The sensitivity of the endogenous luteolytic mechanism was also established by analysing plasma prostaglandin F2alpha metabolite (PGFM) levels following an exogenous oxytocin (50 IU) challenge. The inoculation was given 2 days before a synchronised oestrus and was timed to ensure that viraemia occurred during the initial stage of corpora luteal development. Seven cows inoculated with non-infectious culture medium served as control animals and remained BVDV naive throughout the study. The BVDV challenge was followed by leucopenia, viraemia and sero-conversion. The virus also significantly (P<0.01) reduced plasma oestradiol levels between day 6 and day 11 post-inoculation (i.e. between day 4 and day 9 post-oestrus). However, the infection did not alter (P>0.05) progesterone secretion throughout the oestrous cycle or the plasma concentration of PGFM. These data indicate that bovine follicular cells and oocytes are permissive to BVDV at all stages of follicular development. They also show that a transient fall in oestradiol secretion may accompany an acute infection. In conclusion, this work has identified two potential routes through which BVDV can reduce fertility in the cow, namely impairment of oocyte quality and disruption of gonadal steroidogenesis.     

牛病毒性腹泻病毒NS3蛋白纳米抗体的筛选及反应原性检测

本研究旨在获得高效特异性的牛病毒性腹泻病毒(BVDV)NS3(P80)非结构蛋白的纳米抗体。用BVDV灭活疫苗免疫羊驼,测得抗体效价后分离全血中的淋巴细胞。通过噬菌体展示技术构建羊驼重链抗体可变区噬菌体展示文库。经过连续3次吸附-洗脱-扩增的生物筛淘,从中挑选出与BVDV-NS3蛋白结合的噬菌体。对经菌液PCR、琼脂糖凝胶电泳鉴定到的单域抗体(VHH)克隆进行基因测序和同源性比对。用ELISA方法验证筛选出的纳米抗体的反应原性,找到与BVDV-NS3蛋白亲和力高的纳米抗体。结果表明,获得插入率为92.8%、库容为1.84×1014 CFU/mL的噬菌体展示文库。ELISA结果和氨基酸序列分析显示,成功得到1条与BVDV-NS3蛋白具有良好反应性且与VHH同源性较高的纳米抗体序列。本研究利用大肠杆菌成功表达BVDV-NS3抗原蛋白,建立BVDV纳米抗体噬菌体展示文库,筛选到针对BVDV重要抗原蛋白相应的纳米抗体且与VHH同源性较高。试验结果为牛病毒性腹泻/黏膜病的防控、诊断、治疗及纳米抗体药物的研制提供参考。     

Mapping of two antigenic domains on the NS3 protein of the pestivirus bovine viral diarrhea virus

The immunodominant NS3 (p80) protein of the pestivirus bovine viral diarrhea virus (BVDV) functions as a serine protease and a RNA helicase. To identify antigenic domains of the BVDV NS3, a panel of monoclonal antibodies (mAbs) was tested against fragments of the protein expressed in E. coli. Two large overlapping NS3 fragments, A (amino acids [aa] 1-434) and B (aa 368-683) which together contain all NS3 sequences, were used to screen mAbs for reactivity. Two mAbs, 21.5.8 and 1.11.3, were reactive to fragment A (in ELISA only) and one mAb, 20.10.6, was reactive to fragment B (in ELISA and Western blotting). Further mapping demonstrated that the smallest fragment mAbs 21.5.8 and 1.11.3 bound to was comprised of aa 205-369 (domain A). In Western blotting, the smallest fragment reactive with mAb 20.10.6 was comprised of aa 368-549 (domain B). However, in indirect ELISA, mAb 20.10.6 also demonstrated high reactivity to a smaller fragment comprising aa 368-512 (domain B'). This indicated that the epitope of mAb 20.10.6 was conformational and not linear. Blocking ELISAs using these mAbs and type 1 and type 2 BVDV antisera demonstrated that an immunodominant region of the NS3 protein in cattle is defined by aa 205-549.     

Antigenic characterization of bovine viral diarrhoea virus isolates from Spain with a panel of monoclonal antibodies

A group of 47 bovine viral diarrhoea virus (BVDV) strains isolated from a variety of bovine tissues from eight different geographical areas of Spain and two BVDV strains isolated from a cell line were characterized antigenically with a panel of 23 monoclonal antibodies (mAbs). The mAbs were directed at one of three viral proteins: E2, Erns and NS2-3. A peroxidase-linked assay was used to test the mAbs for reactivity against infected cell monolayers. The data were analysed by two computational methods: the Antigenic Distance Program (MAP) and the Phylogeny Inference Package (PHYLIP), and compared with those obtained previously using the same mAbs with other pestiviruses, including reference strains and UK field isolates. All the Spanish field strains studied appeared to be broadly similar to reference strains of BVDV and were included in the subgroup of classical BVDV, meanwhile the two strains isolated from a cell line were included in the subgroup of atypical pestiviruses.     

Antibody responses against non-structural protein 3 of bovine viral diarrhoea virus in milk and serum samples from animals immunised with an inactivated vaccine

Antibodies against non-structural protein 3 (NS3, p80) of bovine viral diarrhoea virus (BVDV) were determined in milk from cows vaccinated with an inactivated BVDV vaccine and compared to serum antibody levels. Animals in one herd were vaccinated with an inactivated BVDV vaccine according to the standard protocol and animals from a second herd with an intensive schedule. Serum and milk samples were tested for BVDV NS3 antibodies using five commercial ELISAs. With a few exceptions, vaccination according to the standard schedule did not induce BVDV NS3-specific antibodies in serum or milk. However, after vaccination according to the intensive schedule, anti-NS3 antibodies were detected for a short time in serum and, to a lesser extent, in milk. Bulk milk was a suitable substrate for BVDV monitoring of herds vaccinated with the inactivated BVD vaccine.     

Screening and identification of B cell epitopes of structural proteins of foot-and-mouth disease virus serotype Asia1

The objective of this study was to screen and identify the B cell epitopes of structural proteins of foot-and-mouth disease virus (FMDV) serotype Asia1. The complete amino acid sequence of all the four structural proteins (P1 region) was analyzed using the DNAStar Protean system. Seventeen peptides were predicted and selected as potential B cell epitopes. The potential B cell epitope genes were cloned into the pGEX-6P-1 plasmid, then expressed and purified. The resulting 17 glutathione S-transferase (GST) fusion peptides were detected by Western blot and ELISA for evaluation of their antigenicity. Six of the 17 fusion peptides were identified successfully by sera from rabbits immunized with the purified P1 polyprotein of FMDV type Asia1. The six fusion proteins were epi1-1 (VP1:₁TTTTGESADPVT₁₂), epi1-2 (VP1:₁₇NYGGETQTARRLH₂₉), epi1-6 (VP1:₁₉₄TTQDRRKQEIIAPEKQTL₂₁₁), epi2-2 (VP2:₄₀EDAVSGPNTSG₅₀), epi3-1 (VP3:₂₆YGKVSNPPRTSFPG₃₉), and epi4-2 (VP4:₃₀YQNSMDTQLGDN₄₁). The results of this study lay a foundation for further study of the structure and function of the structural proteins and may aid in the design of an epitope vaccine against foot-and-mouth disease (FMD) type Asia1. This study has also shown that the bioinformatics method, in combination with molecular biology methods can be used to map the B cell epitopes on viral proteins.     

Genetic characterization of bovine viral diarrhea virus strains in Beijing,China and innate immune responses of peripheral blood mononuclear cells in persistently infected dairy cattle

To acquire epidemiological data on the bovine viral diarrhea virus (BVDV) and identify cattle persistently infected (PI) with this virus, 4,327 samples from Holstein dairy cows were screened over a four-year period in Beijing, China. Eighteen BVD viruses were isolated, 12 from PI cattle. Based on genetic analysis of their 5''-untranslated region (5''-UTR), the 18 isolates were assigned to subgenotype BVDV-1m, 1a, 1d, 1q, and 1b. To investigate the innate immune responses in the peripheral-blood mononuclear cells of PI cattle, the expression of Toll-like receptors (TLRs), RIG-I-like receptors, interferon-α (IFN-α), IFN-β, myxovirus (influenza virus) resistance 1 (MX1), and interferon stimulatory gene 15 (ISG15) was assessed by qPCR. When compared with healthy cattle, the expression of TLR-7, IFN-α, and IFN-β mRNA was downregulated, but the expression of MX1 and ISG-15 mRNA was upregulated in PI cattle. Immunoblotting analysis revealed that the expression of interferon regulatory factor 3 (IRF-3) and IRF-7 was lower in PI cattle than in healthy cattle. Thus, BVDV-1m and 1a are the predominant subgenotypes in the Beijing region, and the strains are highly divergent. Our findings also suggest that the TLR-7/IRF-7 signaling pathway plays a role in evasion of host restriction by BVDV.     

Genetic analysis of bovine viral diarrhoea viruses from Australia

Eighty-nine bovine viral diarrhoea viruses (BVDV) from Australia have been genetically typed by sequencing of the 5' untranslated region (5'-UTR) and for selected isolates the N(pro) region of the viral genome. Phylogenetic reconstructions indicated that all of the samples examined clustered within the BVDV type 1 genotype. Of the 11 previously described genetic groups of BVDV-1, 87 of the samples examined in this study clustered with the BVDV-1c, while two samples clustered with the BVDV-1a. Based on these analyses there appears to be limited genetic variation within the Australian BVDV field isolates. In addition, the phylogenetic reconstructions indicate that the clustering of Australian BVDV in the phylogenetic trees is not a result of geographic isolation.     

Bovine viral diarrhoea virus (BVDV) subgenotypes in diagnostic laboratory accessions: distribution of BVDV1a, 1b, and 2a subgenotypes

The prevalence of bovine viral diarrhoea virus (BVDV) biotypes and subgenotypes was determined from 131 BVDV positive samples from a diagnostic laboratory. The majority of the isolates were from Oklahoma; however, other states including Kansas, Texas, and Arkansas were represented. These BVDV samples were from submissions of 76 live animals and 55 necropsy samples. There were 131 BVDV samples represented by 117 noncytopathic (NCP), 11 cytopathic (CP) and 3 cases with mixed NCP and CP biotypes. The NCP isolates were more common (P < 0.05) than the CP and NCP/CP combination. The BVDV samples were segregated into three subgenotypes by differential PCR and sequencing of a viral genomic region, 5'-untranslated region (5'-UTR). There were more BVDV1b subgenotypes 60/131 (45.8%) than BVDV1a, 37/131 (28.2%) or BVDV2a, 34/131 (26.0%) (P < 0.05). The organ system involvement included the major categories such as respiratory, digestive, mixed/multiple organs, abortions, and persistent infections (PI). All three BVDV subgenotypes were found in persistently infected (PI) cattle and respiratory diseases, both major requests for BVDV diagnosis. Only one of the 131 viruses was genetically similar to the strains present in U.S. vaccines.