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 short incubation serum neutralization test for bovine viral diarrhea virus.

A three day serum neutralization (SN) test for the detection of antibodies to bovine viral diarrhea virus (BVDV), which is an improvement on the existing five day test, is described. The improved test results in a more rapid viral cytopathic effect and utilizes Madin Darby kidney (MDBK) cells, and horse serum as a medium supplement. A comparison of tests utilizing the NADL and the Singer strains of BVDV and the use of either secondary bovine kidney cells with calf serum (BKCS) or continuous MDBK cells with horse serum (MDHS) was performed. Analysis of the SN results of 685 serum samples from 445 Quebec and Ontario cattle showed that there was no difference, as expected, in the means of the SN antibody titers when the NADL strain was used in either the BKCS or MDHS system but SN antibody titers were elevated (p less than 0.01) when the Singer strain was used in the MDHS system. The SN test with the Singer strain also yielded significantly higher titers for sera from 200 Alberta cattle.     

Inhibition of porcine reproductive and respiratory syndrome virus replication by short hairpin RNA in MARC-145 cells

Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most important contagious agents of swine in the world. The current vaccines cannot provide highly effective protection. In this study, the ability of specific short hairpin RNA directed against different genomic regions of PRRSV to inhibit virus replication in MARC-145 cells was examined. Seven plasmids expressing shRNA targeted to GP5 and nucleocapsid (N) protein coding region of PRRSV S1 strain RNA were constructed and delivered into MARC-145 cells. After infection, these cells, transfected with plasmids pSUPER-N3 or pSUPER-G1, showed a significant decrease in virus yield when compared to control cells, by detection using virus titers (TCID50), indirect immunofluorescence assay and real-time RT-PCR. The antiviral effect was sequence-specific and dose-dependent and could sustain for 96 h. Furthermore, by combination of treatment with plasmid pSUPER-N3 and pSUPER-G1, the viral inhibition cloud be significantly increased. In addition, the viral suppression efficiency by shRNA in previously infected cells was not significant different from that induced by shRNA before viral infection. It indicated that administration of the two different shRNA could have a synergistic effect. RNA interference targeting to the various regions of PRRSV might be a potential alternative virus control strategy.     

牛病毒性腹泻病毒双抗体夹心ELISA检测方法的建立

将牛病毒性腹泻病毒超免疫血清以常规方法提取IgG，采用过碘酸钠法标记辣根过氧化物酶（HRP），建立了从粪样中检测牛病毒性腹泻病毒抗原的双抗体夹心ELISA。结果，抗体的最佳包被量为150μg／mL，酶标抗体最适工作浓度为1：200；封闭液为50mL／L的兔血清；待检粪样及酶标抗体的感作时间为37℃ 120min；底物显色时间为室温15min。应用建立的检测方法对河北省8个大中型奶牛场298份乳牛腹泻粪样进行了检测，结果，阳性检出率为42．6％。     

Response of cattle persistently infected with noncytopathic bovine viral diarrhea virus to vaccination for bovine viral diarrhea and to subsequent challenge exposure with cytopathic bovine viral diarrhea virus

Nine steers persistently infected with noncytopathic bovine viral diarrhea (BVD) virus were allotted into 3 groups (3 cattle/group). Cattle in group A were vaccinated with a modified-live BVD virus vaccine of porcine cell origin, cattle in group B with a modified-live BVD virus vaccine of bovine cell origin, and cattle in group C with a killed BVD virus vaccine of bovine cell origin. Detrimental effects due to vaccination were not seen. Six weeks after vaccination, the steers were challenge exposed with a cytopathic BVD virus. All steers developed mucosal disease after challenge exposure, produced antibodies that neutralized various isolates of BVD virus, and remained persistently infected until death. Steers given killed virus vaccine had a minimal neutralizing-antibody response and developed mucosal disease as quickly as reported for challenge-exposed, nonvaccinated, persistently infected cattle. Steers given modified-live virus vaccines had higher neutralizing-antibody response and longer intervals from challenge exposure to development of mucosal disease. The specificity of the neutralizing-antibody response differed between groups of vaccinated cattle.     

Evaluation of bovine viral diarrhea virus uptake by preimplantation embryos

Bovine embryos were exposed to bovine viral diarrhea (BVD) virus in vitro. An uptake of BVD virus by the embryos could not be detected by several assay systems. A significant decrease in the titer of BVD virus was found to occur when the virus was incubated in saline solution + 5% goat serum or minimal essential medium + 5% goat serum for 24 hours at 37 C. Since there was significant inactivation of the BVD virus during the incubation period, lack of viral infectivity of the embryos may have been due to adverse effects of the experimental environmental conditions on the virus or the embryos or upon viral-embryo interaction.     

Severe clinical disease induced in cattle persistently infected with noncytopathic bovine viral diarrhea virus by superinfection with cytopathic bovine viral diarrhea virus

Eight healthy cattle that were persistently infected with noncytopathic bovine viral diarrhea virus (BVDV) were inoculated with cell culture fluids that contained noncytopathic or cytopathic BVDV. A severe disease occurred after inoculation with cytopathic BVDV. The clinical signs, lesions, and immune response were consistent with those of clinical BVDV infections.     

Structural proteins of bovine viral diarrhea virus.

A procedure for the purification of radioactively labeled bovine viral diarrhea virus was critically evaluated. Purification of virus from artificial mixtures of unlabeled infected and labeled noninfected cells indicated that the extent of purification was approximately 100-fold with respect to host proteins. Residual host proteins were found to contaminate the viral preparation even after extensive purification by differential and isopycnic zonal centrifugation. Co-electrophoresis of 3H-labeled virus with 14C-labeled host cell material in neutral sodium dodecyl sulfate-7.5% polyacrylamide gels provided a means to distinguish viral specific proteins from host cell protein contaminants. Four major electrophoretic components were identified as being of viral origin; molecular weights of the components were estimated from their migration rates relative to protein markers of known molecular weight. Two viral components (VC), VC 1 and VC 3, migrated heterogeneously and had molecular weights of 93,000 to 110,000 and 50,000 to 59,000 daltons, respectively. Molecular weights of VC 2 and VC 4 were 70,000 and 25,000 daltons, respectively.     

Evolution of bovine viral diarrhea virus vaccines.

Control of bovine viral diarrhea virus (BVDV) infection is economically important to the cattle industry because the virus causes a variety of clinical diseases that adversely affect essentially all stages of the production cycle. Production losses primarily stem from reproductive failure and from immunosuppression during acute BVDV infection, which predisposes calves to respiratory or enteric diseases. Control is achieved by implementing herd health pro-grams focused on limiting exposure by avoiding persistently infected (PI) carrier cattle and by optimizing protective immunity through immunization. Vaccination cannot be relied upon solely to protect against fetal infection and losses due to BVD. This is because no single BVDV vaccine has been shown to give complete fetal protection. In addition to strategic use of vaccines, herd management practices should also be implemented to identify and eliminate PI carrier cattle and to avoid exposure to BVDV infection.     

牛病毒性腹泻病毒和牛轮状病毒TaqMan二重实时荧光RT-PCR检测方法的建立

根据牛病毒性腹泻病毒（BVDV）5′端非编码区和牛轮状病毒（BRV）VP6基因序列,设计特异性引物和探针。通过对引物和探针浓度、Mg2＋浓度、dNTP浓度和Taq酶用量以及反应条件等因素的优化筛选,建立了能同时鉴别BVDV和BRV的二重荧光RT-PCR方法。该方法特异性好,与其他病原如CSFV、MB和IBRV不发生交叉反应;敏感性高,能够检测100个BVDV RNA和100个BRV RNA;稳定性好,批内重复和批间重复变异系数小;干扰性试验表明该方法能同时检测2个模板的不同浓度组合。本研究建立的二重荧光RT-PCR方法可用于BVDV和BRV检测,具有特异、敏感、快速、稳定等优点,是BVDV和BRV基础研究、流行病学调查和临床检测的良好工具。     

Biosecurity and biocontainment of bovine viral diarrhea virus.

Infection of cattle with BVDV results in a variety of clinical illnesses costly to the cattle industry worldwide. The reservoir and primary source of transmission is cattle born PI with BVDV after transplacental infection in early gestation. It is a challenge to determine with certainty whether or not BVDV is circulating among a herd of cattle. If the virus is present in a herd,then biocontainment strategies are used to eliminate the virus by testing to removing PI cattle, preventing exposure of pregnant cattle to the virus, and increasing resistance to infection using vaccination. If it is clear that the virus is not present in a herd then, biosecurity actions must be taken to prevent introducing the virus into the herd.     

二重RT-PCR同时检测VSV与BVDV核酸

水泡性口炎病毒(VSV)与牛病毒性腹泻病毒(BVDV)具有相近的传播途径与类似的检测方法，本文参照文献报道的基因序列，设计合成了两对能分别扩增VSV(202bp)、BVDV(341bp)基因片段的引物，并对PCR扩增条件进行优化，建立了二重RT-PCR方法，可同时检测VSV与BVDV病毒核酸。VSV产物经测序显示与报道的核酸序列同源性为88．6％。二重RT-PCR同时检测VSV与BVDV经济、快速、敏感、特异，可用于实验研究和流行病学调查。     

Monitoring bovine viral diarrhea virus vaccines for adventitious virus, using T1 ribonuclease viral RNA oligonucleotide fingerprinting.

Viral RNA oligonucleotide fingerprinting was used to discriminate 3 cytopathic vaccine bovine viral diarrhea viruses (BVDV) grown in medium supplemented with serum contaminated with noncytopathic BVDV from the same 3 viruses grown in cell culture free of BVDV. Oligonucleotide fingerprinting also effectively discriminated between reference Singer BVDV, NADL BVDV, and New York-1 BVDV grown in BVDV-free noncontaminated or BVDV-contaminated cell cultures. Oligonucleotide fingerprint mapping of viral RNA maybe used to determine the purity of virus stocks, as well as that of BVDV vaccines.     

Differentiation of cytopathic and noncytopathic isolates of bovine viral diarrhea virus by virus neutralization

Soluble antigens of cytopathic and noncytopathic isolates of bovine viral diarrhea virus were resolved by high-performance liquid gel-permeation chromatography into 4 major and 3 minor peaks. The 2 peaks with the larger molecular weights (240,000 and 140,000 daltons) were immunogenic when inoculated into rabbits. Virus neutralizing antibodies were specific for the homologous virus. The soluble antigens were determined to be greater than 100,000 daltons by filtration.     

牛病毒性腹泻病毒人工感染绵羊病理学研究

牛病毒性腹泻病毒(BVDV)属于黄病毒科、瘟疫病毒属的代表种,病毒基因组为单链正股RNA,长约12.5 kb.该病毒在自然条件下,牛易感,但羊、猪、鹿、驼等其他野生动物也可感染,近年来已有人感染BVDV的报道[1].     

牛病毒性腹泻疫苗的研究进展

牛病毒性腹泻(bovine viral diarrhea,BVD)作为一种病毒性腹泻黏膜病,给养殖业造成了巨大的经济损失,应用疫苗来防控牛病毒性腹泻病毒(bovine viral diarrhea virus,BVDV)依然是重要的方法。直至目前,我国还没有成熟的BVDV疫苗,本研究主要综述了近几年来国外BVDV疫苗的研究进展,旨在为国内BVDV疫苗的研制及应用提供参考。     

Nature of early reproductive failure caused by bovine viral diarrhea virus

A 2-part study was undertaken to determine the effect of bovine viral diarrhea (BVD) virus on fertilization and early development of embryos. In experiment 1, 10 seronegative cows were superovulated and artificially inseminated twice during estrus. After the second insemination, 5 of the cows received intrauterine infusion of BVD virus suspension. The other 5 cows received suspending medium only and served as controls. All 10 cows were slaughtered on day 3, and ova and embryos were collected for morphologic evaluation. A total of 49 and 52 ova and embryos were collected from the control and virus-treated cows, respectively. Among the ova and embryos collected from control cows, 81.6% were fertilized, whereas only 52% were fertilized in the virus-treated group. The statistically significant difference (P less than 0.01) indicated that the virus interferes with fertilization. In experiment 2, the protocol was identical except for slaughter on day 13. Seventy-nine ova and embryos were collected from the 6 control cows, and the 6 virus-treated cows yielded 59 ova and embryos. Of the total ova and embryos recovered on day 13, 88.6% and 50.8% were hatched and developing normally in the control and virus-treated groups, respectively. The difference was highly significant (P less than 0.001). Unfertilized ova and degenerating embryos could not be differentiated on the basis of morphologic appearance. The nearly identical percentages of unfertilized ova in experiment 1 and unhatched ova and embryos in experiment 2 strongly suggested that fertilization failure is the principal manifestation of the observed adverse effect of BVD virus infection.     

Efficacy of bovine viral diarrhea vaccine used in Japan against bovine viral diarrhea virus type 2 strain 890

Bovine viral diarrhea virus (BVDV) has been segregated into two genotypes, type 1 and type 2. To determine the efficacy of the commercially available bovine viral diarrhea type 1 vaccine used in Japan against BVDV type 2, calves were infected with BVDV type 2 strain 890 4 weeks after administration of the vaccine. The vaccinated calves did not develop any clinical signs and hematological changes such as observed in unvaccinated calves after the challenge. Furthermore, the challenge virus was not recovered from the vaccinated calves throughout the duration of the experiment, whereas it was recovered from all unvaccinated calves. The bovine viral diarrhea vaccine used in Japan is efficacious against infection with BVDV type 2 strain 890.     

Methods for detection and frequency of contamination of fetal calf serum with bovine viral diarrhea virus and antibodies against bovine viral diarrhea virus.

Methods used by the National Animal Disease Center to test fetal calf serum for contamination with bovine viral diarrhea virus (BVDV) and antibodies against BVDV are described. Using those methods, virus was isolated from 332 of 1,608 (20.6%) lots of raw fetal calf serum obtained specifically for the Center and 93 of 190 (49%) lots of commercially available fetal calf serum. Virus neutralization and immunoperoxidase staining tests were used to detect antibodies against BVDV in 224 of the 1,608 (13.9%) lots of raw fetal calf serum. Both BVDV and antibodies against BVDV were detected in 50 lots of raw serum. The molecular specificity of antibodies against BVDV was determined by radioimmunoprecipitation. Lots of fetal calf serum that contained BVDV-specific antibodies that did not neutralize virus were identified.