利用RNAi抑制口蹄疫病毒的复制

根据口蹄疫病毒 IRES和 L 串联序列两侧的保守区域设计了 2个引物 ,利用 RT- PCR和 PCR方法扩增出该串联序列 ,并进行了测序。测序结果表明 ,扩增产物与 Gen Bank上相应的序列具有很高的序列同源性 (大于 99% )。在测序的基础上 ,选择了 L 基因上的 1个靶位点 (位于启始密码子下游第 2 2 9nt后 2 1 nt长的序列 ) ,合成了 si RNA表达盒SEC- L2 2 9。细胞单层长成 5 0 %～ 70 %时 ,将纯化的 SEC- L2 2 9转染到 BHK细胞中 ,转染 4 h后用高感染复数 FMDV接种 ,2 4 h后用间接免疫荧光方法对口蹄疫病毒在 BHK细胞中的复制进行检测。研究结果表明 ,SEC- L 2 2 9极大地抑制了口蹄疫病毒在 BHK细胞中的复制 ,且该抑制作用具有序列特异性 ,并降低了 BHK细胞的死亡率。另外 ,2 5 ng和5 0 ng SEC- L2 2 9处理组间对病毒复制的抑制作用差异不明显 ,可能是病毒基因组发生了突变。本试验表明 ,利用 PCR方法合成的 SEC在 BHK细胞中能特异性地抑制 FMDV的复制 ,RNAi技术可能为防治口蹄疫提供一个新的途径     

An investigation into causes of resistance of a cloned line of BHK cells to a strain of foot-and-mouth disease virus

The reduced ability of foot-and-mouth disease virus (FMDV) strain Asia 1 Iran 1/73 to replicate in the cloned BHK cell line AA7 was not due to lack of virus attachment at the cell surface. Instead, the main restriction in the viral growth cycle occurred during synthesis and processing of viral macromolecules, and/or during the earliest stages of their assembly. Reduced efficiency of penetration and uncoating of virus attached to the cells may also have contributed to inhibition of virus replication. Viral components or subviral particles did not accumulate and defective interfering particles were not detected. The reduced number of infective virions produced was released from infected cells at the normal rate. No interferon production could be demonstrated.     

犬呼肠病毒3型在BHK-21细胞上增殖的研究

采用酶标ＳＰＡ法进行了犬呼肠３ 型病毒在ＢＨＫ２１ 细胞上定位的研究。结果表明：接毒后６ ｈ 即可在胞浆见到絮状阳性反应,２４ ｈ 后呈强阳性着染;Ｈ．Ｅ．染色在胞浆内可见强嗜酸性着染或嗜酸性包涵体,提示可能是病毒感染引起的特征性病变,感染后细胞病变发展较快,６０ ｈ 细胞大部脱落;病毒血凝滴度结果也证实了细胞病变与病毒增殖有关。     

光生物素标记的口蹄疫病毒cDNA探针的制备及其对口蹄疫病毒RNA检测的试验研究

本文首次报道了利用pF1034质粒制备FMDV O型特异性探针,并通过PCR反应扩增FMDO_1K株病毒基因组的第2962位与3071位之间共110bp序列,制备了能检测O型、A型和亚洲I型FMDV RNA的群(组)特异性探针。用硝酸纤维素膜斑点杂交试验表明,二者均能检测出10pg水平的O_9K毒株的纯RNA;但前者只与O型FMDV RNA杂交,与A型及亚洲I型FMDV RNA无交叉杂交现象;而后者则能与O型、A型和亚洲I型的FMDV RNA发生杂交反应。对照试验显示:此两种探针与SVDV ssRNA、BTV dsRNA、EHDV dsRNA、DHV ssRNA、PRV DNA、乳鼠组织细胞RNA、BHK_(21)克隆13细胞RNA及DNA等均不出现交叉杂交现象,但与IBR DNA有假阳性杂交反应。     

口蹄疫疫苗的悬浮培养工艺研究

通过生物反应器中进行BHK21细胞悬浮培养并逐级放大,分别接种口蹄疫OJMS/2000株与Asia 1/JSL株,纯化灭活后制备50批疫苗,结果均符合《口蹄疫O型、亚洲Ⅰ型二价疫苗(OJMS株+ JSL株)制造及检验规程》（以下称规程）所规定的各项标准,病毒146S抗原含量比转瓶培养提高10倍以上、疫苗的不良反应得到进一步改善.     

悬浮培养在口蹄疫疫苗中的应用

通过生物反应器中进行BHK21细胞悬浮培养并逐级放大,分别接种口蹄疫OJMS/2000株与Asia 1/JSL株,纯化灭活后制备50批疫苗,结果均符合《口蹄疫O型、亚洲I型二价疫苗（OJMS株＋JSL株）制造及检验规程》（以下称规程）所规定的各项标准,病毒146S抗原含量比转瓶培养提高10倍以上、疫苗的不良反应得到进一步改善。     

口蹄疫病毒3C蛋白酶结构与功能及应用研究进展

口蹄疫病毒(FMDV)3C蛋白酶是FMDV基因组编码中具有酶学活性的病毒产物之一,在FMDV编码蛋白的成熟和子代病毒在宿主细胞体内大量扩增中发挥着重要作用。3C蛋白酶能剪切多聚蛋白,降解特定的蛋白质,是宿主细胞中重要的毒力因子。3C蛋白酶能调控蛋白的转录和翻译,使宿主细胞内的干扰素等多种抗病毒基因低水平表达,使FMDV逃避宿主的天然免疫。论文主要综述了FMDV 3C蛋白酶的结构、生物学功能,并介绍了其在研制新型疫苗中的应用,以期为今后FMDV 3C蛋白酶的研究、新型疫苗的研发提供参考。     

The plasmid constructs producing shRNA corresponding to the conserved 3D polymerase of Foot and Mouth Disease virus protects guinea pigs against challenge virus

RNA interference (RNAi) has been used as an effective antiviral strategy for its specific silencing of viral gene expression in mammalian cells. In this study, shRNA targeting two regions of Foot and Mouth Disease Virus (FMDV) i.e. 3D and 5'UTR which are very essential in virus replication were evaluated. The constructs were made using h7K RNA polymerase III promoter. We investigated in vivo inhibitory effect of shRNA on FMDV replication in BHK-21 cells and guinea pigs. The results showed that transfection of 3D shRNA could reduce virus growth by three folds when cells were challenged with 10(2) TCID(50) of FMDV. Pretreated guinea pigs with 3DshRNA were protected 80% with 10(3) GPID(50) of FMDV. As a first report in guinea pigs which are recognized animal model for FMD vaccine potency testing, the study suggests that shRNA could be a viable therapeutic approach to control severity of FMD infection and spread.     

Molecular characterization of a 13-amino acid deletion in VP1 (1D) protein and novel amino acid substitutions in 3D polymerase protein of foot and mouth disease virus subtype A/Iran87

The nucleotide sequence of the VP1 (1D) and partial 3D polymerase (3D^pol) coding regions of the foot and mouth disease virus (FMDV) vaccine strain A/Iran87, a highly passaged isolate (~150 passages), was determined and aligned with previously published FMDV serotype A sequences. Overall analysis of the amino acid substitutions revealed that the partial 3D^pol coding region contained four amino acid alterations. Amino acid sequence comparison of the VP1 coding region of the field isolates revealed deletions in the highly passaged Iranian isolate (A/Iran87). The prominent G-H loop of the FMDV VP1 protein contains the conserved arginine-glycine-aspartic acid (RGD) tripeptide, which is a well-known ligand for a specific cell surface integrin. Despite losing the RGD sequence of the VP1 protein and an Asp₂₆→Glu substitution in a beta sheet located within a small groove of the 3D^pol protein, the virus grew in BHK 21 suspension cell cultures. Since this strain has been used as a vaccine strain, it may be inferred that the RGD deletion has no critical role in virus attachment to the cell during the initiation of infection. It is probable that this FMDV subtype can utilize other pathways for cell attachment.     

Asia I型口蹄疫疫苗效力检验替代方法的初步研究

使用三株针对AsiaⅠ型口蹄疫146S抗原的杂交瘤细胞株(1#G3C2、2#G6B9、10#C2G1)大量生产腹水单克隆抗体,粗提纯化并按一定的比例进行混合。初步建立混合腹水单克隆抗体介导的间接夹心ELISA方法,使用该方法测定AsiaⅠ型病毒灭活抗原、O型病毒灭活抗原、正常BHK21细胞培养液,结果表明该方法特异性好,所测定OD值和AsiaⅠ型抗原稀释倍数呈明显线性关系。用该方法对11批AsiaⅠ型、O型单价或双价成品疫苗破乳抗原进行测定,结果表明不同批次不同企业生产的疫苗完整病毒颗粒抗原含量有差异,进一步完善有望用于AsiaⅠ型口蹄疫成品疫苗中有效颗粒抗原含量的测定,从而发展成一种疫苗效力检验替代方法。     

无血清悬浮培养BHK21细胞增殖伪狂犬病毒的参数研究

旨在筛选适合生物反应器悬浮培养BHK21细胞的无血清培养基,并利用该技术培养伪狂犬病毒。通过筛选市售的无血清培养基,以细胞形态、细胞活率和增殖倍数作为驯化指标,对BHK21细胞进行无血清驯化;对使用10 L和40 L生物反应器无血清悬浮培养BHK21细胞的参数进行研究;同时利用筛选的无血清培养基和悬浮细胞进行伪狂犬病毒增殖测试。结果表明,市售培养基Ⅰ符合筛选要求,其培养的细胞形态比较均一,结团少,细胞生长2 d的增殖倍数为3.5倍,细胞活率在95%以上;生物反应器悬浮培养BHK21细胞的最适参数为转速80 r/min、pH值7.2、DO 60%;利用该工艺技术培养伪狂犬病毒,当接种细胞密度为6×10⁶ cells/mL,接毒剂量为0.3%时,48 h收获的病毒液滴度可达10^9.5 TCID₅₀/mL。该试验实现了伪狂犬病毒的无血清悬浮生产,为其生产工艺的优化提供了可能。     

Genome analysis and development of infectious cDNA clone of a virulence-attenuated strain of foot-and-mouth disease virus type Asia 1 from China

The RNA genome sequence of the rabbit passage-attenuated strain of foot-and-mouth disease virus (FMDV) Asia 1, ZB/CHA/58(att), was determined to be 8165 nt in length excluding the poly(C) tract in the 5′ UTR and the poly(A) tail at the 3′ end. ZB/CHA/58(att) was most similar to the vaccine strain Asia 1/YNBS/58 in genome sequence and there were no deletions or insertions within the deduced polyprotein between ZB/CHA/58(att) and YNBS/58, but there were a total of 25 substitutions at the amino acid level and an extra 19-nt stretch in the 5′ UTR was found in ZB/CHA/58(att). An infectious full-length cDNA clone of ZB/CHA/58(att) was developed. Infectious virus could be recovered in BHK-21 cells transfected with the synthetic viral RNA transcribed in vitro. The plaque morphology, growth kinetics and antigenic profile of the infectious clone-derived virus (termed tZB) were indistinguishable from those induced by the parental virus. Furthermore, the virulence properties of ZB/CHA/58(att) and tZB were found to be highly similar in the mouse model. The availability of genome sequence information and infectious cDNA clone of the FMDV ZB/CHA/58(att) lays a new ground for further investigation of FMDV virulence determinants and development of new potent vaccine to FMD.     

Immunogenicity of foot-and-mouth disease virus type O1 replicated in either monolayer or suspended BHK cell system

The efficacy of vaccines formulated from the 10th passage of foot-and-mouth disease virus (FMDV) type O1 in monolayer baby hamster kidney (BHK) cells and the 8th passage in suspension BHK cells was compared in steers. The vaccines were inactivated with ethylenimine, contained an equal amount of antigen and were emulsified in oil-adjuvant. Six animals were vaccinated with each vaccine. During the challenge of immunity (91 days post-vaccination, DPV), one out of the six steers from the monolayer vaccine group became infected with the challenge virus while none of the six steers from the suspension vaccine group contracted the disease during the test period. The neutralizing antibody titers (means) of the serum samples taken at different DPV also did not suggest significant variation between these vaccines. In addition exposure to FMDV infected animals demonstrated that both vaccines elicited an immune state in the vaccinates.     

Protection of cattle and swine against foot-and-mouth disease, using biosynthetic peptide vaccines

A single dose of foot-and-mouth disease (FMD) virus protein 1 (VP1) peptide, expressed in Escherichia coli as a fusion protein with 190 amino acids (AA) of the LE' protein of the tryptophan operon of E coli, elicited an immune response in steers sufficient to withstand the challenge of exposure to animals with acute FMD. The 58-micrograms dose of viral peptide, composed of a segment of the VP1 from the A12 strain (A12) of FMD virus (FMDV; A12-32dimer) in a tandem repeat configuration of AA137 through 168 and emulsified with oil adjuvant, elicited a serologic response in cattle equivalent to that obtained using conventional whole virus vaccines. Two groups of swine were vaccinated, 1 with the A12-32dimer as used in cattle and 1 with AA131 through 157 from VP1 of the A24 strain (A24) of FMDV (A24-peptide), expressed in the same system as A12-32dimer, but as a single copy per molecule. In swine, the 58-micrograms dose of the A12-32dimer repeated at 28 days was an effective immunogen; all swine were protected against A12 and, in addition, the vaccine protected 50% of the swine against A24. The 29-micrograms dose of A24-peptide, administered according to the same schedule, elicited protection against A24 in 50% of the vaccinates and, in addition, protected 25% of those vaccinates against A12. The serologic response elicited by A12-32dimer against A24 virus was considerably greater than the response elicited by A24-peptide against A12 virus. The evidence of multiple immunogenic epitopes between AA131 and AA168 was evaluated.     

Rapid methodology for antigenic profiling of FMDV field strains and for the control of identity, purity and viral integrity in commercial virus vaccines using monoclonal antibodies

Monoclonal antibodies (MAbs) developed against different foot-and-mouth disease virus (FMDV) vaccine strains were extensively used to study any possible antigenic variations during vaccine production in Argentine facilities. Additionally, a typing ELISA using strain specific MAbs was developed to detect potential cross contaminations among FMDV strains in master and working seeds with high specificity and sensitivity and to confirm strains identity in formulated vaccines. This assay was carried out for the South American strains currently in use in production facilities in Argentina (A24/Cruzeiro, A/Argentina/01, O1/Campos and C3/Indaial) and for the strain O/Taiwan, produced only for export to Asia. These non-cross reactive MAbs were also used to analyze the integrity of viral particles belonging to each one of the individual strains, following isolation of 140S virions by means of sucrose density gradients from the aqueous phase of commercial polyvalent vaccines. Antigenic profiles were defined for FMDV reference strains using panels of MAbs, and a coefficient of correlation of reactivity with these panels was calculated to establish consistent identity upon serial passages of master and production seeds. A comparison of vaccine and field strain antigenic profiles performed using coefficients of correlation allowed the rapid identification of two main groups of serotype A viruses collected during the last FMD epidemic in Argentina, whose reactivity matched closely to A/Argentina/2000 and A/Argentina/2001 strains.     

口蹄疫病毒多基因及猪α干扰素共表达载体的构建及其免疫豚鼠效果研究

为探讨口蹄疫病毒多基因及猪α干扰素（IFN-α）基因共表达真核质粒进入临床试验的可行性,本试验用PCR方法扩增了口蹄疫病毒P12A3C及部分2B基因（P12X3C）和猪IFN-α基因,克隆到真核表达载体pBudCE4.1中,经双酶切鉴定后,将重组质粒pBudCE4.1-P12X3C-IFN-α转染BHK-21细胞中,观察目的基因的表达,并将重组质粒免疫豚鼠,检测豚鼠的血清抗体水平、中和抗体滴度及T淋巴细胞增殖情况。结果显示,经酶切鉴定及DNA序列分析成功构建了重组质粒pBudCE4.1-P12X3C-IFN-α,转染BHK-21细胞后,通过Western blotting、间接免疫荧光试验鉴定证实重组质粒能有效表达。ELISA结果显示,重组质粒pBudCE4.1-P12X3C-IFN-α比重组质粒pBudCE4.1-P12X3C能诱导机体产生更高水平的抗口蹄疫病毒的血清抗体,且中和抗体滴度也高于重组质粒pBudCE4.1-P12X3C组。MTT法检测结果表明,重组质粒pBudCE4.1-P12X3C-IFN-α组淋巴细胞增殖可达15%,而重组质粒pBudCE4.1-P12X3C组则为11%。攻毒后重组质粒pBudCE4.1-P12X3C-IFN-α组和灭活疫苗组保护率达100%,高于重组质粒pBudCE4.1-P12X3C组的80%。本试验成功构建了重组质粒pBudCE4.1-P12X3C-IFN-α,猪IFN-α作为佐剂可有效辅助口蹄疫DNA疫苗提高动物体内的免疫反应。     

Direct contact transmission of three different foot-and-mouth disease virus strains in swine demonstrates important strain-specific differences

A novel direct contact transmission model for the study of foot-and-mouth disease virus (FMDV) infection of swine was utilized to investigate transmission characteristics of three FMDV strains belonging to serotypes A, O and Asia1. Each strain demonstrated distinct transmission characteristics and required different exposure times to achieve successful contact transmission. While a 4h exposure was sufficient for strain A24 Cruzeiro (A24Cru), both O1 Manisa and Asia1 Shamir transmission required 18h or more. Viral excretion levels from donors (for all three strains) and virus present in room air (for A24Cru and O1 Manisa) were evaluated and associated with clinical signs and observed transmission pattern. Although all directly inoculated donor animals showed acute FMD, A24Cru had the highest levels of viral shedding in saliva and nasal swabs followed by O1 Manisa and Asia1 Shamir. Virus levels in room air were higher and were detected longer for A24Cru than for O1 Manisa. These results provide direct evidence for important strain-specific variation in transmission characteristics and emphasize the need for thorough evaluation of different FMDV viral strains using a well defined contact transmission methodology. This information is critical for vaccine and biotherapeutic efficacy testing, pathogenesis and disease modeling of FMDV transmission.     

Attenuated Salmonella choleraesuis-mediated RNAi targeted to conserved regions against foot-and-mouth disease virus in guinea pigs and swine

In this study, specific sequences within three genes (3D, VP4 and 2B) of the foot-and-mouth disease virus (FMDV) genome were determined to be effective RNAi targets. These sequences are highly conserved among different serotype viruses based on sequence analysis. Small interfering RNA (siRNA)-expressing plasmids (p3D-NT19, p3D-NT56, pVP4-NT19, pVP4-NT65 and p2B-NT25) were constructed to express siRNA targeting 3D, VP4 and 2B, respectively. The antiviral potential of these siRNA for various FMDV isolates was investigated in baby hamster kidney (BHK-21) cells and suckling mice. The results show that these siRNA inhibited virus yield 10- to 300-fold for different FMDV isolates of serotype O and serotype Asia I at 48 h post infection in BHK-21 cells compared to control cells. In suckling mice, p3D-NT56 and p2B-NT25 delayed the death of mice. Twenty percent to 40% of the animals that received a single siRNA dose survived 5 days post infection with serotype O or serotype Asia I. We used an attenuated Salmonella choleraesuis (C500) vaccine strain, to carry the plasmid that expresses siRNA directed against the polymerase gene 3D (p3D-NT56) of FMDV. We used guinea pigs to evaluate the inhibitory effects of recombinant S. cho (p3D-NT56/S. cho) on FMDV infection. The results show that 80% of guinea pigs inoculated with 10⁹ CFU of p3D-NT56/S. cho and challenged 36 h later with 50 ID₅₀ of homologous FMDV were protected. We also measured the antiviral activity of p3D-NT56/S. cho in swine. The results indicate that 100% of the animals treated with 5 × 10⁹ CFU of p3D-NT56/S. cho were protected in 9 days.     

Attenuation of turkey meningo-encephalitis virus in BHK21 cells

Turkey meningo-encephalitis virus was adapted to BHK21 cell culture. Cytopathic effects were characterized by rounding and detachment of cells within 48 hours. Attenuation was achieved by 41 successive passages in BHK21 cell cultures. Turkeys and Japanese quail (Coturnix coturnix japonica), kept under laboratory conditions and inoculated with the attenuated virus, did not develop symptoms of turkey meningo-encephalitis but reacted by the production of haemagglutination inhibition antibody. They resisted intracerebral challenge with pathogenic strains of turkey meningo-encephalitis virus.     

Foot-and-mouth disease virus (FMDV) experimental infection: susceptibility and immune response of adult mice

Adult mice are susceptible to foot-and-mouth disease virus (FMDV) infection only under some experimental conditions. This paper report the results of pathogenesis studies on 4 different strains of mice (CF1, C3H, NIH-nude, BALB-c/J) infected with the cloned and uncloned 0(1)C strain of FMDV. High virus titers were detected in blood and pancreas 12-24 h after infection (p.i.); these persisted for up to 48 h p.i. in CF1 and BALB-c/J mice and 72 h p.i. in the two other mouse strains. Virus titers observed in other organs were lower than those found in blood. In pancreas, and occasionally in salivary glands, oropharynx, heart and testicles, viral antigen was detected by direct immunofluorescent assay. Circulating neutralizing antibodies appeared in CF1 and C3H mice at 72 and 96 h p.i. respectively, and their titers remained unchanged during the 30-day experimental period. Antibodies against viral infection-associated antigen (VIA) were detected for a shorter period. In animals irradiated with 1 LD 50 (total body irradiation), viremia persisted up to 14 days p.i. and a low antibody response was observed which began at the end of viremia. No differences in the response of mice to cloned or uncloned FMDV were observed.