Enzyme-Linked Immunosorbent Assays Using the Recombinant gp51 and p24 of Bovine Leukemia Virus for Immunodetection of the Disease

Enzyme-linked immunosorbent assay (ELISA) is often used to test bovine leukemia virus (BLV) infection. However, commercially available kits test in South America detect only antibodies against the gp51 protein. With the aim to improve the sensitivity of the test, we developed here a two-step indirect dual ELISA test that included both proteins p24 and gp51, expressed and produced in E. coli and baculovirus expression system respectively. Two hundred ten BLV sera, stated as double positive or double negative by the combination of commercial agar gel immunodiffusion (AGID) assay and a gp51-ELISA test, were tested with our in house dual rp24/rgp51 ELISA. Firstly, we checked the purified, optimized and standardized proteins as antigen by the checkerboard technique, and set up our in house ELISA test. The concordance correlation coefficient (CCC) and coefficient of variation (CV) intraplate repeatability levels were within the values established by the international standards. The statistical analysis demonstrated the value of sera correctly ranked highest (93.48%), and for 0.3 cutoff, the sensitivity was 95.65% and the specificity 91.30%. In conclusion, the rp24/rgp51 ELISA developed and standardized here demonstrated to have good analytical characteristics to be considered for screening of BLV.

Enhanced Disinfection Effect of a Compound Disinfectant against Bovine Herpes Virus Type 1 at Low Temperature

A compound disinfectant, which consisted of didecyldimethylammonium chloride (DDAC), ortho-dichlorobenzene (1,2-dichlorobenzene, ODB), and chlorocresol (4-chloro-3-methylphenol, CC), and its component chemicals were individually tested for effectiveness against bovine herpes virus type 1 (BHV-1). DDAC, DDAC+ODB, DDAC+CC, and DDAC+ODB+CC showed effectiveness against BHV-1 at room temperature. However, ODB, CC, and ODB+CC showed no virucidal effects. The effects of all disinfectants tested were decreased at low temperature. DDAC showed disinfectant effects at a dilution of 1/800 and DDAC+ODB, DDAC+CC, and DDAC+ODB+CC at dilutions of 1/800 and 1/1600 at low temperature in the presence of 2% fetal bovine serum (FBS) but ODB, CC, and ODB+CC showed no virucidal effects. At low temperature and in the presence of 10% FBS, DDAC, DDAC+ODB, and DDAC+CC showed disinfectant effects at dilutions of 1/800, whereas ODB, CC, and ODB+CC showed no virucidal effects. DDAC+ ODB+CC was more effective (at 1/800 and 1/1600) than the other disinfectants under these conditions. In conclusion, a combination of three disinfectant components (DDAC+ODB+CC), enhanced the disinfectant effects at low temperature and in organic matter contamination.

An Experimental Model for Screening Anti-AIDS Drugs with Bovine Immunodeficiency Virus

本文应用牛免疫缺陷病毒（ＢＩＶ）合胞体形成和ＢＩＶ长末端重复序列（ＬＴＲ）引导的萤火虫荧光素酶（Ｌｕｃ）基因表达分析，建立了抗艾滋病（ＡＩＤＳ）药物的筛选和评价模型。ＢＩＶ合胞体形成及ＢＩＶ－ＬＴＲ引导的Ｌｕｃ基因活力与ＢＩＶ感染的细胞呈线性关系。临床有效药物３′叠氮胸嘧啶脱氧核苷（ＡＺＴ）可显著抑制ＢＩＶ合胞体的形成及ＢＩＶ－ＬＴＲ引导的Ｌｕｃ基因表大家水平，并有剂量依赖关系。上述两种分得到ＡＺＴ的ＩＣ５０分别为０．２４和０．０５２ｍｍｏｌ／Ｌ。

Characterization of a full-length infectious clone of bovine foamy virus 3026

The biological features of most foamy viruses(FVs) are poorly understood, including bovine foamy virus(BFV). BFV strain 3026(BFV3026) was isolated from the peripheral blood mononuclear cells of an infected cow in Zhangjiakou, China. A full-length genomic clone of BFV3026 was obtained from BFV3026-infected cells, and it exhibited more than 99% amino acid(AA) homology to another BFV strain isolated in the USA. Upon transfection into fetal canine thymus cells, the full-length BFV3026 clone produced viral structural and auxiliary proteins, typical cytopathic effects, and virus particles. These results demonstrate that the full-length BFV3026 clone is fully infectious and can be used in further BFV3026 research.

Seroprevalence of Bovine Parainfluenza Virus Type 3 (bPI-3V) in Ruminants from Grenada

Respiratory viral infections are known for serious economic losses in ruminants. Bovine parain-fluenza 3 virus (bPI-3V) a member of Respirovirus genus in association with other respiratory viruses causes respiratory disease complex in ruminants. The aim of this study was to estimate the seroprevalence of bPI-3V in non vaccinated cattle, sheep and goats from Grenada. Sera were collected randomly from 60 sheep, 60 goats and 60 cattle from all six parishes of Grenada. Sera were tested for antibodies to bPI-3V using an indirect Enzyme Linked Immunosorbant Assay (ELISA) kit. Antibodies to bPI-3V were detected in 13.4% (Confidence Level (CL): 95%;Confidence Interval (CI): 4.76% to 22.02%) in cattle;16.7% (CL: 95%;CI: 7.26% to 26.14%) in sheep and 11.7% (CL: 95%;CI: 3.57% to 19.83%) in goats. There was statistically no significant difference in prevalence (p > 0.05) of antibodies to bPI-3V in cattle, sheep and goats in Grenada. This is the first report on seroprevalence of bPI-3V in ruminants in Grenada, West Indies.

A New Indicator Cell Line Established to Monitor Bovine Foamy Virus Infection

In order to improve the accuracy for quantitating the bovine foamy virus (BFV) in vitro,we developed a baby hamster kidney cell (BHK)-21-derived indicator cell line containing a plasmid that encodes the firefly luciferase driven by the BFV long terminal repeat promoter (LTR,from-7 to 1012).The BFV titer could be determined by detecting the luciferase expression since the viral trans-activator BTas protein activates the promoter activity of the LTR.One clone,designated BFVL,was selected from ten neomycin-resistant clones.BFVL showed a specific and inducible dose-and time-dependent luciferase activity in response to BFV infection.Although the changes in luciferase activity of BFVL peaked at 84 h post infection,it was possible to differentiate infected and uninfected cells at 48 h post infection.A linear relationship was established between the multiplicity of infection (MOI) of BFV and the activated ratio of luciferase expression in BFVL.Moreover,the sensitivity of the BFVL-based assay for detecting infectious BFV was 10,000 times higher than the conventional CPE-based assay at 48 h post infection.These findings suggest that the BFVL-based assay is rapid,easy,sensitive,quantitative and specific for detection of BFV infection.

Effects of Matrine on Bovine Papilloma Virus-infected C127 Cells

The aim of this study was to select drugs for prevention and treatment of bovine papilloma in vitro, using bovine papilloma virus（BPV-2） infected mouse mammary epithelial（C127） cell lines as the infectious cell model system. C127 cell shapes changed with BPV infection observed under light microscopy. C127 cell shapes changed with BPV infection observed under light microscopy. PCR was used to confirm BPV infected C127 cells. mR NA levels of matrine to BPV-2 infected C127 cells were detected by RT-PCR. Apoptosis of BPV-infected C127 cells treated with matrine was analyzed by flow cytometry. Results showed that 80 μg · mL-1 matrine had a direct virucidal and intervention effect of BPV-2. Matrine inhibited the proliferation of BPV-infected cells. Therefore, matrine might be regarded as a therapeutic drug candidate for BPV induced bovine papilloma disease treatment in future.

Bovine Leukemia Virus Gene Segment Detected in Human Breast Tissue

Background: Bovine Leukemia Virus (BLV) is known by infections in bovine cattle and produce, in 30% of infected animals, persistent lymphocytosis significantly impacts the beef industry. It has been proposed that this virus could be transmitted to humans and be present in cases of breast cancer. Aim: to determine the presence of 380 bp of gag gene segment of BLV in paraffin-embedded breast tissue. Study Design: Control-case study. Methodology: 106 tissue samples were collected. 53 were cancer positive samples and 53 were negative samples for this pathology. After dewaxing tissues, DNA was extracted, amplified and sequenced. Phylogenetic analysis was done in order to verify BLV gene segment, presence and origin. Results: 43 samples were positive (40.5%) for BLV segment. In the case group this segment was found in 35.8% of the samples and in the control group, BLV presented in 45.2% of the samples. Phylogenetic analysis confirmed BLV presence and had shown a high homology between amplified gene sequences obtained from human breast tissues and those coming from bovine cattle with leukosis reported by GenBank. Conclusion: The presence of BLV genes in humans and its location in breast tissue can be confirmed, however, it should be clarified as a possible promoter of malignancy processes on this tissue.

A Quantitative Assay for Measuring of Bovine Immunodeficiency Virus Using a Luciferase-based Indicator Cell Line

In order to quantitate the bovine immunodeficiency virus (BIV) infection in vitro, a BIV indicator cell line (BIVL) was established by transfecting baby hamster kidney cells with reporter plasmids containing the firefly luciferase gene driven by a BIV long terminal repeat promoter. The BIV activates promoter activity of the LTR to express luciferase upon infection. BIV infection could therefore by quantified by detection of luciferase activity. Compared to standard assays used to detect BIV infection, the BIVL-based assay is 10 times more sensitive than the the CPE-based assay, and has similar sensitivity with the viral capsid protein Western blot assay. BIV indicator cell line could detect BIV infection specifically. Luciferase activity of BIV infected BIVL cells showed a time dependent manner, and 60 h post infection is the optimal time to detect BIV infection. Luciferase activity of BIVL cells correlates with the BIV capsid protein expression. Moreover, a linear relationship was found between MOI and the activated intensity of luciferase expression. In brief, the BIV indicator cell line is an easy, robust and quantitive method for monitoring BIV infection.

Evaluation of an attenuated vaccine candidate based on the genotype C of bovine parainfluenza virus type 3 in albino guinea pigs

Bovine parainfluenza virus type 3（BPIV3） is considered as one of the most important respiratory tract pathogens of both young and adult cattle, and widespread among cattle in the world. BPIV3 was first reported in China in 2008 and four strains of BPIV3 were isolated from Shandong Province, known as genotype C（BPIV3c）. Pathogen investigations had shown that BPIV3 c infection was very common among cattle in China. To date, BPIV3 can be classified into genotypes A, B and C based on genetic and phylogenetic analysis. Serological survey also demonstrates that BPIV3 infection is widespread in China, however, there is still no available vaccine for BPIV3 prevention in China nowadays. In the present study, the BPIV3 c strain SD0835 was continuously passaged on Madin-Darby bovine kidney（MDBK） cells for hundreds of times, and the pathogenicity of passage 209 was reduced in guinea pigs. The passage 209 of BPIV3 c strain SD0835 was used as a live vaccine candidate to immunize the guinea pigs. The vaccination results revealed that two vaccinations could induce excellent serum neutralizing antibody responses as well as proliferation of T lymphocytes. The vaccinated guinea pigs were well protected against challenge with a low passage of BPIV3 c strain SD0835. Additionally, the percentages of CD4~＋ and CD8~＋ T cell subsets of animals in vaccinated group increased after immunization; T cell subsets on day 2 after challenge in both groups decreased, and the decline of CD4~＋ and CD8~＋ T cell subsets levels of four guinea pigs in vaccinated group was relatively moderate, comparing with that of the control group. These data support further testing of the attenuated virus as an effective candidate vaccine.

Construction and characterization of chimeric BHIV (BIV/HIV-1) viruses carrying the bovine immunodeficiency virus gag gene

AIM: To explore the possibility of the replacement of the gag gene between human immunodeficiency virus and bovine immunodeficiency virus, to achieve chimeric virions,and thereby gain a new kind of AIDS vaccine based on BHIV chimeric viruses.METHODS: A series of chimeric BHIV proviral DNAs differing in the replacement regions in gag gene were constructed, and then were transfected into 293T cells. The expression of chimeric viral genes was detected at the RNA and protein level. The supematant of 293T cell was ultra centrifuged to detect the probable chimeric virion. Once the chimeric virion was detected, its biological activities were also assayed by infecting HIV-sensitive MT4 cells.RESULTS: Four chimeric BHIV proviral DNAs were constructed. Genes in chimeric viruses expressed correctly in transfected 293T cells. All four constructs assembled chimeric virions with different degrees of efficiency. These virions had complete structures common to retroviruses and packaged genomic RNAs, but the cleavages of the precursor Gag proteins were abnormal to some extent. Three of these virions tested could attach and enter into MT4 cells, and one of them could complete the course of reverse transcription. Yet none of them could replicate in MT4 cells.CONCLUSION: The replacement of partial gag gene of HIV with BIV gaggene is feasible. Genes in chimeric BHIVs are accurately expressed, and virions are assembled. These chimeric BHIVs (proviral DNA together with virus particles) have the potential to become a new kind of HIV/AIDS vaccine.

Subcellular Localization Analysis of Bovine Foamy Virus Borf1 Protein

The Borf1 protein is encoded by an immediate-early gene of the bovine foamy virus (BFV) and plays a key role in the viral life cycle. Borf1 is a DNA binding protein which can transactivate both the long terminal repeat (LTR) and the internal promoter (IP) of BFV by specifically binding to the transactivation responsive element (TRE). To analyze the subcellular localization of Borf1 during the BFV life cycle, this gene was cloned into a prokaryotic expression vector and expressed in a soluble form. After the purification and immunization, we raised the mouse anti-Borf1 serum with a high titer based on ELISA results. Western blot analysis showed that the antiserum could specifically recognize the Borf1 protein that was expressed in 293T cells. With this specific serum, we revealed the nuclear and cytoplasmic localization of Borf1 in HeLa cells that was transfected with Borf1. Moreover, the immuno-fluorescence assay also showed that the localization of Borf1 during the infection and transfection of BFV was identical.

Expression and Antigenic Characterization of the Epitope-G_1 of the Bovine Ephemeral Fever Virus Glycoprotein in Pichia pastoris

The epitope-G1 gene of Bovine ephemeral fever virus(BEFV) glycoprotein was synthesised by PCR and cloned into expression vector pPIC9K to construct recombinant plasmid pPIC9K-G1.Then the pPIC9K-G1 was linearized and transformed into Pichia pastoris GS115.The recombinant P.pastoris strains were selected by a G418 transformation screen and confirmed by PCR.After being induced with methanol,an expressed protein with 26 kDa molecular weight was obtained,which was much bigger than the predicted size(15.54 kDa).Deglycosylation analysis indicated the recombinant G1 was glycosylated.Western blot and ELISA tests,as well as rabbit immunization and specificity experiments indicated that the target protein had both higher reaction activity and higher immunocompetence and specificity.The recombinant G1 protein could be used as a coating antigen to develop an ELISA kit for bovine ephemeral fever diagnosis.

Prevalence of coronavirus from diarrheic calves in the Republic of Korea

Objective: To investigate the prevalence of bovine coronavirus(BCo V), bovine rotavirus, and bovine viral diarrhea virus in the feces of normal and diarrheic Korean native calves aged 1-81 days between April and October of 2016 in the Republic of Korea. Methods: Samples were obtained from 50 normal and 93 diarrheic(56 semi-formed, 28 loose, and 9 watery feces) calves in six different regions of northern and southern Korea. These fecal samples were tested for BCo V, bovine rotavirus, and bovine viral diarrhea virus by RT-PCR. Results: Among the three pathogens examined, infection with BCo V was especially prominent in relation to diarrhea among calves aged 1-21 days [odds ratio(OR)=9.3, 95% confidence interval(CI): 1.1-78.9; P=0.02). Infection with BCo V alone(OR=2.9; 95% CI: 1.1-7.6; P=0.03) or coinfection of BCo V with bovine viral diarrhea virus(OR=3.6; 95% CI: 1.0-12.4; P=0.04) was significantly associated with the development of loose feces. Grazing and colostrum intake strongly reduced the occurrence of diarrhea as compared to housed calves(OR=0.2; 95% CI: 0.1-0.4; P=0.00) and calves that had not been fed colostrum(OR=0.2; 95% CI: 0.1-0.7; P=0.02), respectively. Conclusions: The present study suggests that BCo V is involved in calf diarrhea in the Republic of Korea. Therefore, grazing and colostrum intake is recommended for preventing and controlling calf diarrhea caused by BCoV.

An Indirect ELISA of Classical Swine Fever Virus Based on Quadruple Antigenic Epitope Peptide Expressed in E.coli

In this study,a synthesized quadruple antigenic epitope gene region of the classical swine fever virus (CSFV)E2 glycoprotein was expressed in E.coli to a obtain target protein.This target protein was used as a coating antigen to establish an indirect ELISA for specifically detecting anti-CSFV antibodies in serum samples from pigs.The P/N cut-off value of this assay was 1.92 by receiver operating characteristic curve(ROC)analysis based on 30 negative sera and 80 positive samples.The test gave 97.5%sensitivity and 96.7%specificity compared with the indirect hemagglutination(IHA)test.The inter-assay and intra-assay coefficients of variation (CVs)for 16 sera were both≤6.8%.No cross-reactivity between the coating antigen and anti-bovine viral diarrhoea virus(BVDV)antibodies was observed.

Brazilian vaccinia virus strains show a classical orthopoxvirus infection course and cross-protection

【正】Objectives:The purpose of this work was to study the infection course and cross-protection in mice after intradermal injection of Vaccinia virus(VACV) strain Western Reserve and three Brazilian VACV strains: Aracatuba,Muriae and BeAn58058 isolated from cow,human and rodent,respectively.Methods:Balb/c mice were inoculated by footpad and back scarification and daily monitored regarding lesion development and weight loss.To check cross protection after intradermal VACV inoculation,mice were subsequendy infected with different VACV strains and monitored to check lesion development.Serum neutralization assays were performed to check for the presence of antibodies against Orthopoxvirus.Results:After VACV intradermal inoculation the lesion development pattern was similar in mice infected with the different virus strains.By using the footpad scarification model,cross-protection among VACV strains was observed.Moreover,neutralizing antibodies against Orthopoxvirus were detected in sera from mice infected with all VACV strains.Conclusion:Although it was not possible to observe virulence differences among VACV strains isolated from cow,rodent and human using the murine model,this inoculation route showed to be an appropriated model to study lesions development since it mimics natural infections by VACV in nature.

牛病毒性腹泻病毒E2蛋白的真核表达及间接ELISA抗体检测方法的建立

为建立检测牛病毒性腹泻病毒(Bovine viral diarrhea virus,BVDV)抗体的间接ELISA方法,利用昆虫细胞真核表达系统成功表达E2蛋白,将纯化后的E2蛋白作为包被抗原,用方阵滴定方法对影响ELISA的各个因素进行优化,并进行特异性、敏感性和重复性试验。结果表明,在昆虫细胞中表达了BVDV E2蛋白,Western blot证实目的蛋白可与BVDV阳性血清发生特异性反应。ELISA优化结果显示,E2蛋白最佳包被浓度为0.5μg/mL,最佳封闭液为1%明胶,最佳血清稀释度为1∶400,最佳血清作用方式为37℃作用30 min,酶标抗体的最佳作用方式为1∶2000稀释、37℃作用30 min,最佳底物作用时间为室温20 min,阳性临界值为OD 450≥0.423。与血清中和试验法进行比较,总符合率为97.8%,板内和板间重复性试验的变异系数均小于10%。该方法与牛常见病毒阳性血清均无交叉反应。说明建立的间接ELISA抗体检测方法特异性、敏感性和重复性良好,可用于大批量样本的临床检测和流行病学研究。

健康安格斯犊牛与IBRV感染犊牛鼻腔菌群变化比较

牛传染性鼻气管炎病毒(IBRV)是一种对全球养牛业造成严重影响的牛呼吸系统病毒。对新疆南疆地区4个规模化安格斯肉牛繁育场1月龄安格斯犊牛进行IBRV感染流行病学调查,探讨IBRV感染犊牛的鼻腔菌群变化。临床调查出现呼吸道疾病症状(主要包括发热、咳嗽、呼吸困难)的1月龄安格斯犊牛,采集犊牛鼻拭子,进行IBRV PCR检测,依据PCR检测结果,随机选取单纯IBRV阳性犊牛(P组)和IBRV阴性且无其他呼吸道病毒感染的健康犊牛(N组)各10头,采用16S rRNA基因测序技术,选择V3和V4可变区使用Illumina平台对鼻腔菌群DNA片段进行双端(Paired-end)测序,分析两组犊牛鼻腔菌群组成结构和功能预测。结果显示,该牛场犊牛出现呼吸道疾病症状共计922头,犊牛呼吸道疾病临床症状发生率为8.2%(922/11 215);其中死亡98头,病死率为10.6%(98/922)。样品IBRV检出率为22.0%(50/227)。与N组犊牛鼻腔菌群分类单元数相比,P组犊牛在门、纲、目、科水平上呈极显著增加(P<0.01),且属水平也呈增加趋势(P=0.056)。Alpha多样性显示,P组犊牛鼻腔菌群均匀度(Pielou_e)和覆盖度(Goods_coverage)指数显著高于N组犊牛(P<0.05),Beta多样性中P组犊牛鼻腔菌群结构与N组犊牛有显著差异(P<0.05)。菌门和菌属差异性显示,P组犊牛的厚壁菌门(Firmicutes)丰度极显著低于N组犊牛(P<0.01),绿弯菌门、酸杆菌门(Acidobacteria)、蓝菌门(Cyanobacteria)、芽单胞菌门(Gemmatimonadetes)丰度极显著高于N组犊牛(P<0.01);P组犊牛的动性球菌属(Planococcus)、盐水球菌属(Salinicoccus)丰度显著低于N组犊牛(P<0.05),嗜盐单胞菌属(Halomonas)、乳酸杆菌属(Lactobacillus)丰度显著高于N组犊牛(P<0.05)。P组犊牛在MetaCyc代谢通路中存在9条代谢通路变化,在KEGG代谢通路丰度预测中存在7条代谢通路变化,主要和参与合成,炎性反应标志物和影响机体生长发育相关。此外,两组间鼻腔菌群在细胞功能、物质运输、分解和合成代谢以及疾病发生等预测功能方面也存在差异。本研究初步揭示了呼吸道症状病牛群中IBRV感染和鼻腔菌群组成结构及功能变化密切相关,为进一步探明犊牛感染IBRV后的发病机制提供了理论参考。

不同基因型牛病毒性腹泻病毒对BALB/c小鼠致病性研究

牛病毒性腹泻病毒(BVDV)是危害养牛业的重要病原。2021年本实验室从患有肺炎的牛肺中分离出1株BVDV(毒株编号为JS2201),经鉴定为1b亚型。为了评价该分离株的毒力与致病性,分别建立了分离株与参考株BVDV-1型Oregon C24V株、BVDV-2型C1602株BVDV感染BALB/c小鼠模型,通过检测攻毒后小鼠临床症状、体重变化、病毒血症情况、血液中白细胞与淋巴细胞的变化、组织器官的病理组织学变化及病毒载量,比较了分离株JS2201与1型和2型参考株的致病性差异。结果显示:BVDV分离株JS2201与参考株均能够成功感染BALB/c小鼠,攻毒BALB/c小鼠均形成病毒血症,导致血液中白细胞数量、淋巴细胞数量以及淋巴细胞百分比短暂下降;BVDV分离株JS2201株攻毒组小鼠肝脏、肾脏、肺脏、脾脏中BVDV载量要高于C24V、C1602株攻毒组;攻毒组小鼠均表现为间质性肺炎病变,JS2201株攻毒组与1型参考株C24V株攻毒组小鼠肺脏病理组织学变化相似,BVDV-2型参考株C1602株攻毒组小鼠肺脏病变更为严重。综上,本研究成功建立了不同亚型BVDV急性感染BALB/c小鼠的模型,且比较分析了分离株与参考株之间的致病性差异,为BVDV疫苗的研制以及致病机制的研究奠定了基础。