绵羊痘病毒和山羊痘病毒双重PCR检测方法的建立及应用

为建立鉴别绵羊痘病毒(SPPV)和山羊痘病毒(GTPV)的检测方法,本研究针对这2种病毒的基因组序列,分别设计2对特异性引物,通过对引物浓度、退火温度等的优化,建立了快速鉴别检测SPPV和GTPV的双重PCR方法。该方法分别扩增出SPPV长度为177 bp和GTPV长度为222 bp的目的片段。特异性试验结果显示,该方法对牛疙瘩皮肤病病毒、犬细小病毒、大肠杆菌O157、沙门氏菌、健康羊组织和牛组织均无扩增。敏感性试验显示,该方法最低可检测1.725×107copies/μL的SPPV和1.71×106copies/μL的GTPV。应用该方法对50份临床病料样品进行检测的结果与病毒分离鉴定结果一致,均检出5份感染GTPV的病料和2份感染SPPV的病料,表明该方法可以用于临床病料样品的检测。     

牛结节性皮肤病病毒实验室检测方法研究进展

牛结节性皮肤病(LSD)是由牛结节性皮肤病病毒(LSDV)引起的以发热、消瘦、皮肤水肿、局部形成坚硬的结节或溃疡、淋巴结肿大等为主要特征的急性、亚急性传染病。本病目前尚无特效治疗药物,疫苗接种是当前主要的防控措施,但我国目前并没有针对该病的疫苗,因此建立快速准确的检测方法显得尤为重要。本文从该病的病毒分离和鉴定、血清学及分子生物学等方面对LSDV的最新实验室检测方法研究进展进行了综述,以期为LSD的诊断和防控提供参考。     

Immunohistochemical evaluation of inflammatory infiltrate in the skin and lung of lambs naturally infected with sheeppox virus

The present study describes immunophenotypic characteristics of inflammatory infiltrate in the skin and lung of lambs naturally infected with sheeppox virus (SPV). Three lambs revealed typical cutaneous and pulmonary lesions of sheeppox. Histologically, cutaneous and pulmonary lesions consisted of hyperplastic and/or degenerative changes in the epithelium with mononuclear cells, neutrophils, and typical sheeppox cells (SPCs), which had a vacuolated nucleus and marginated chromatin with occasional granular intracytoplasmic inclusions. The inflammatory infiltrate in pox lesions in both skin and lung was characterized by the presence of MHC II+ dendritic cells, CD4+, CD8+, gammadelta+ T cells, IgM+ cells, and CD21+ cells. Loss of expression of MHC I and MHC II antigens was observed in the affected areas of skin and lung. SPCs, stained with anti-SPV antibody, were also positive for CD14 and CD172A, antigens expressed on monocytes and macrophages. CD14 and CD172A negative SPCs were considered to be SPV infected degenerated epithelial cells or fibroblasts.     

Recombinant Kluyveromyces lactis expressing highly pathogenic porcine reproductive and respiratory syndrome virus GP5 elicits mucosal and cell-mediated immune responses in mice

Currently, killed-virus and modified-live porcine reproductive and respiratory syndrome virus (PRRSV) vaccines are used to control porcine reproductive and respiratory syndrome. However, both types of vaccines have inherent drawbacks; accordingly, the development of novel PRRSV vaccines is urgently needed. Previous studies have suggested that yeast possesses adjuvant activities, and it has been used as an expression vehicle to elicit immune responses to foreign antigens. In this report, recombinant Kluyveromyces lactis expressing GP5 of HP-PRRSV (Yeast-GP5) was generated and immune responses to this construct were analyzed in mice. Intestinal mucosal PRRSV-specific sIgA antibody and higher levels of IFN-γ in spleen CD4⁺ and CD8⁺ T cells were induced by oral administration of Yeast-GP5. Additionally, Yeast-GP5 administered subcutaneously evoked vigorous cell-mediated immunity, and PRRSV-specific lymphocyte proliferation and IFN-γ secretion were detected in the splenocytes of mice. These results suggest that Yeast-GP5 has the potential for use as a vaccine for PRRSV in the future.     

Viruses as vectors

Traditional vaccines against diseases caused by viruses are based on live attenuated viruses or killed virus preparations. Through the application of molecular biology it is now possible to consider several new approaches to making vaccines, which may combine increased efficacy with greater safety. One of these approaches is to manipulate genetically a virus so that it carries and expresses a foreign gene (or part of a gene) which codes for a protective antigen for another disease. Adeno-, polio- and herpesviruses have been engineered to act as vectors in this way but vaccinia virus remains the main candidate for a recombinant virus vector for vaccine use. The broad host-range of vaccinia virus has made it an effective vector for the analysis of expression of "foreign" antigens as well as a tool for the dissection of the host animal's immune system. For practical purposes in veterinary vaccines, recombinant viruses based on other poxviruses, with more restricted host-ranges, may have certain advantages. Work on the development of recombinant avipoxviruses and capripoxviruses as prototype vaccines for use in poultry and ruminants, respectively, is discussed and illustrated.     

A virus vector based on Canine Herpesvirus for vaccine applications in canids

Canine Herpesvirus (CHV) is being developed as a virus vector for the vaccination of European red foxes. However, initial studies using recombinant CHV vaccines in foxes revealed viral attenuation and lack of antibody response to inserted foreign antigens. These findings were attributed both to inactivation of the thymidine kinase (TK) gene and excess foreign genetic material in the recombinant viral genome. In this study, we report an improved CHV-bacterial artificial chromosome (BAC) vector system designed to overcome attenuation in foxes. A non-essential region was identified in the CHV genome as an alternative insertion site for foreign genes. Replacement of a guanine/cytosine (GC)-rich intergenic region between UL21 and UL22 of CHV with a marker gene did not change growth behaviour in vitro, showing that this region is not essential for virus growth in cell culture. We subsequently produced a CHV-BAC vector with an intact TK gene in which the bacterial genes and the antigen expression cassette were inserted into this GC-rich locus. Unlike earlier constructs, the new CHV-BAC allowed self-excision of the bacterial genes via homologous recombination after transfection of BACs into cell culture. The BAC-CHV system was used to produce a recombinant virus that constitutively expressed porcine zona pellucida subunit C protein between the UL21 and UL22 genes of CHV. Complete self-excision of the bacterial genes from CHV was achieved within one round of replication whilst retaining antigen gene expression.     

Risk assessments of lumpy skin diseases in Borena bull market chain and its implication for livelihoods and international trade

Risks of introduction of lumpy skin disease (LSD) through traded Borena bulls to market chain and its consequences were assessed. The assessment used the framework that has been recommended by the World Animal Health Organization (OIE) for risk analysis. Likelihoods for release and exposure were estimated by a qualitative scale ranging from negligible to very high, whereas the consequences which resulted from disease occurrences were assessed quantitatively. The likelihood of the introduction of LSD to the market chain through traded Borena bulls is found to be high (medium uncertainty), whereas the probability of exposure is very high (medium uncertainty). From the total of 11,189 bulls observed during outbreak investigation of LSD in six sites of feedlot operation in and around Adama, 681(6.1 %) and 204 (1.8 %) bulls were found to be affected and dead with LSD, respectively. The total economic loss due to LSD was estimated to be 667,785.6 USD. The risk estimates for LSD are greater than negligible; therefore, disease prevention and control strategy along the chain should be carefully considered by the Ethiopian veterinary services.     

牛结节性皮肤病及其病原检测方法研究进展

牛结节性皮肤病(LSD)是由牛结节性皮肤病病毒(LSDV)引起的一种传染病.该病起源于非洲,在撒哈拉和马达加斯加呈地方流行,欧美国家鲜有发生,我国境内尚无牛结节性皮肤病.在疫病流行的非洲等地区和国家,该病的检测技术主要有病原分离鉴定、常规血清学检测和分子生物学检测.对于没有该病发生的地区和国家,有学者应用有交叉反应的同...     

表达红色荧光蛋白的重组鸭肠炎病毒的构建及鉴定

鸭肠炎病毒(Duck enteritis virus, DEV),又称为鸭瘟病毒,是一种只感染雁形目禽类的疱疹病毒。DEV具有基因组大、非必需基因多、能插入外源基因的容量大、遗传稳定等优点,其庞大而复杂的基因组为外源基因提供了诸多可插入位点,以DEV为载体,成功表达了禽流感病毒HA蛋白_^[1]、鸭病毒性肝炎病毒VP0蛋白_^[2]、鸭坦布苏病毒E基因_^[3]以及鹅细小病毒VP2蛋白_^[4]。构建重组DEV的重要一步是将报告基因插入到基因组中,目前常用的报告基因有绿色荧光蛋白(GFP)、增强型绿色荧光蛋白(EGFP)、红色荧光蛋白(RFP)以及LacZ报告基团。本研究用RFP报告基团插入DEV UL2基因中,获得表达红色荧光的重组病毒,一步生长曲线表明,RFP对DEV的生长无影响;连续传代12代,RFP能够稳定表达,为DEV载体研究奠定基础。     

牛结节性皮肤病病毒研究进展

牛结节性皮肤病(LSD)在全世界范围内均有流行和记载,对我国养牛业构成了严重的威胁。接种疫苗是目前防控LSDV最有效的方法,其中减毒活疫苗在许多国家均有使用。近年研究表明,LSDV活疫苗的使用会造成疫苗重组病毒的流行,接种后会引起动物不同程度的不良反应。对LSDV的病原学特征、分离鉴定方法、流行病学的情况、检测方法以及LSDV疫苗的研究进展进行了阐述,以期为牛结节性皮肤病的免疫预防以及鉴别诊断提供研究思路。     

表达绿色荧光蛋白的MVA重组毒株的构建及筛选

为了构建及筛选表达绿色荧光蛋白（GFP）的改良型痘苗病毒安卡拉（MVA）重组毒株,并对其进行鉴定,本研究基于同源重组原理设计引物,通过PCR扩增获取含有MVA两侧同源臂的外源基因GFP片段,将GFP基因片段转染到感染了MVA的CEF细胞中使之同源重组到MVA ORF086-087位点（基因组中70303-70304 bp之间）,利用倒置荧光显微镜观察并标记表达GFP的单个噬斑,筛选获取重组毒株,应用倒置荧光技术、PCR及Western blotting对该重组毒株进行鉴定。结果显示,经过3轮噬斑筛选,在倒置荧光显微镜下可观察到大量表达GFP的单个噬斑,PCR扩增检测结果表明目的基因已成功整合到重组毒株MVA-GFP中。Western blotting结果表明,GFP在感染的细胞内成功表达。本研究利用基因工程技术成功获得表达GFP的重组毒株MVA-GFP,可为进一步将其他抗原基因插入GFP位点中筛选无标记的重组毒株及疫苗研究提供材料。     

一起境外输入性牛结节性皮肤病疫情

2020年11月，山东滨州市、东营市引进牛皮肤出现自限性皮肤结节、损伤以及结痂等症状，疑似发生牛结节性皮肤病（lumpy skin disease，LSD）。为确诊两地疫情及了解病原的遗传演化关系，利用荧光定量PCR方法进行诊断，以PCR方法扩增GCPR基因并进行核苷酸比对分析和遗传演化分析。检测结果显示，采集的样品中检测到牛结节性皮肤病病毒（lumpy skin disease virus，LSDV），两地疫情确诊为LSD疫情。GCPR基因核苷酸比对结果显示，China/SDBinzhou/2020、China/SDDongying/2020 GCPR基因存在12个核苷酸的插入，与疫苗毒株Neethling vaccine LW 1959株、Neethling-LSD vaccine-OBP株以及俄罗斯发现的疫苗样毒株Saratov株在GCPR基因的核苷酸插入序列相同，具备疫苗样毒株的特征。系统发育分析结果表明，China/SDBinzhou/2020、China/SDDongying/2020 GCPR基因与我国新疆发现的LSDV毒株GCPR基因处同一小分支中，亲缘关系较近。同时，我国LSDV毒株与Neethling vaccine LW 1959株、Neethling-LSD vaccine-OBP株以及Saratov株等共处于一大分支中。综上，确诊滨州市、东营市两地疫情为LSD疫情，这是山东省首次确诊输入性LSD疫情。     

Use of the Capripoxvirus homologue of Vaccinia virus 30 kDa RNA polymerase subunit (RPO30) gene as a novel diagnostic and genotyping target: development of a classical PCR method to differentiate Goat poxvirus from Sheep poxvirus

Sheep poxvirus (SPPV), Goat poxvirus (GTPV) and Lumpy skin disease virus (LSDV) are Capripoxviruses (CaPVs) responsible for causing severe poxvirus disease in sheep, goats and cattle, respectively. Serological differentiation of CaPVs is not possible and strain identification has relied on the implicitly accepted hypothesis that the viruses show well defined host specificity. However, it is now known that cross infections can occur and authentication of identity based on the host animal species from which the strain was first isolated, is not valid and should be replaced with molecular techniques to allow unequivocal strain differentiation. To identify a diagnostic target for strain genotyping, the CaPV homologue of the Vaccinia virus E4L gene which encodes the 30 kDa DNA-dependent RNA polymerase subunit, RPO30 was analyzed. Forty-six isolates from different hosts and geographical origins were included. Most CaPVs fit into one of the three different groups according to their host origins: the SPPV, the GTPV and the LSDV group. A unique 21-nucleotide deletion was found in all SPPV isolates which was exploited to develop a RPO30-based classical PCR test to differentiate SPPV from GTPV that will allow rapid differential diagnosis of disease during CaPV outbreaks in small ruminants.     

Characterization of serum lysosomal enzymatic activities. II. Effect of lumpy skin disease in Egyptian cows.

Clinical findings and lysosomal enzymes (LYE) in eight lumpy skin diseases (LSD) cows and same number of healthy ones were reported in Tal-El Baker village and Tal Alkabir centre, Ismailia province, Egypt. LSD began with fever, anorexia, skin lesions in form of nodules all over the body, which disappeared spontaneously or gathered to form large lumps. It was complicated with respiratory manifestation, corneal opacity, mastitis, dehydration and later on recumbency. It is noteworthy that the level of 3 LYE showed the same trend of significant reduction in acute stage of the disease (5 days after occurrence of LSD) probably due to injection of animals with a therapeutics dose of terramycin. Acid-phosphatase (ACP) enzyme is the sole that behaved very high significant increase in the serum in acute stage of LSD due to the damaged tissues caused by the virus. It underwent insignificant decrease in late stage of the disease (20 days after its occurrence) to restore the normal LYE level in control cows indicating recovery. Alpha-galactosidase (alpha-GAL) decreased perpetually by the progression of LSD because of the decreased bactericidal index which ist in concomitance with the secondary bacterial invader. N-acetyl-beta-glucosaminidase (beta-NAG) and beta-galactosidase (beta-GAL) in LYE had the same fluctuating manner. The activities showed very highly significant decrease in acute stage, followed by highly significant and significant increases (late LSD stage) respectively. The appreciable significant increase of beta-GAL may declare the effect of anorexia on LSD. In view of these findings, it can be postulated that LSD may be diagnosed and prognosed through LYE changes in the serum.     

Lumpy skin disease in southern Africa: a review of the disease and aspects of control

This article reviews some of the important aspects of lumpy skin disease (LSD) that may impact on its successful control. A resurgence of the disease in the last decade has highlighted some constraints of the Neethling strain vaccine, but there is no evidence of vaccine breakdowns owing to the presence of heterologous field strains. More research is needed on epidemiology and transmission of LSD in South Africa to formulate control measures.     

Efficacy of a BVDV subunit vaccine produced in alfalfa transgenic plants

Bovine viral diarrhea virus (BVDV) is considered an important cause of economic loss within bovine herds worldwide. In Argentina, only the use of inactivated vaccines is allowed, however, the efficacy of inactivated BVDV vaccines is variable due to its low immunogenicity. The use of recombinant subunit vaccines has been proposed as an alternative to overcome this difficulty. Different studies on protection against BVDV infection have focused the E2 protein, supporting its putative use in subunit vaccines. Utilization of transgenic plants expressing recombinant antigens for the formulation of experimental vaccines represents an innovative and cost effective alternative to the classical fermentation systems.The aim of this work was to develop transgenic alfalfa plants (Medicago sativa, L.) expressing a truncated version of the structural protein E2 from BVDV fused to a molecule named APCH, that target to antigen presenting cells (APCH-tE2). The concentration of recombinant APCH-tE2 in alfalfa leaves was 1 μg/g at fresh weight and its expression remained stable after vegetative propagation. A methodology based an aqueous two phases system was standardized for concentration and partial purification of APCH-tE2 from alfalfa. Guinea pigs parentally immunized with leaf extracts developed high titers of neutralizing antibodies. In bovine, the APCH-tE2 subunit vaccine was able to induce BVDV-specific neutralizing antibodies. After challenge, bovines inoculated with 3 μg of APCH-tE2 produced in alfalfa transgenic plants showed complete virological protection.     

Sites of Persistence of Lumpy Skin Disease Virus in the Genital Tract of Experimentally Infected Bulls

The objectives of this work were to determine the site of persistence of lumpy skin disease virus (LSDV) in bulls shedding the virus in semen for a period longer than 28 days, to determine if the virus is present in all fractions of semen and to study lesions that developed in the genital tract. Six serologically negative postpubertal bulls were experimentally infected with a virulent field isolate of LSDV. The polymerase chain reaction (PCR) was performed on sheath washes, vesicular fluid, supernatant and cell‐rich fractions of semen from day 10 to day 26 postinfection (p.i.). Bulls that were positive by PCR on the whole semen sample collected on day 28 p.i. were slaughtered and tissue samples from their genital tracts submitted for histopathological evaluation, immunoperoxidase staining, virus isolation and PCR. Two of the bulls developed severe lumpy skin disease (LSD) and were found to be shedding viral DNA in their semen on day 28 p.i. Viral DNA was identified in all semen fractions from all bulls, but mostly from the cell‐rich fraction and from the severely affected bulls. The PCR assay was positive on postmortem samples of testes and epididymides from the two severely affected bulls. Virus could be recovered from the testes of these two bulls and from the epididymis of one of them. Immunoperoxidase staining was positive for LSDV staining in sections of testes and epididymides exhibiting necrosis. This study suggests that the testis and epididymis are sites of persistence of LSDV in bulls shedding virus in semen for prolonged periods and revealed that viral DNA is present in all fractions of the ejaculate.     

牛结节性皮肤病疫苗研究进展

牛结节性皮肤病可对病畜皮肤造成永久性损害，并会导致产奶量下降、生长发育迟缓、公畜不育和孕畜流产等严重后果，对经济生产具有重要影响。目前尚无治疗该病的特效药，使用安全有效的疫苗是防控该病传播的重要手段。牛结节性皮肤病弱毒活疫苗在许多国家已被成功用于该病的防控，除此之外，由于牛结节性皮肤病病毒与山羊痘病毒及绵羊痘病毒在基因组序列上的高度相似性，山羊痘病毒弱毒活疫苗和绵羊痘病毒弱毒活疫苗在许多地区也被用于该病的防控。牛结节性皮肤病病毒编码基因较多，目前尚未发现基因缺失疫苗和亚单位疫苗具有好的保护效果。近年来灭活疫苗在实验室研究中取得了较大进展，但仍需进一步大规模临床试验以证明其有效性。此外，牛结节性皮肤病病毒作为一种痘病毒，已被证明是表达多种病原微生物抗原的有效载体之一。笔者主要总结了当前牛结节性皮肤病疫苗的研究进展，对弱毒疫苗、灭活疫苗、基因缺失疫苗和亚单位疫苗的使用情况、保护效果及研究方向等方面进行了综述，以期为牛结节性皮肤病疫苗的研发和疫病的防控提供新的思路和见解。     

Alternative cell lines for the propagation of lumpy skin disease virus

In our Institute lumpy skin disease virus is grown on primary lamb testis cells for isolation, identification and vaccine production. However, the availability of lambs in Kenya has been seriously reduced over the past few years. This has led to an increase in the cost of using primary lamb testis cells. This study was undertaken to investigate other primary cell lines, which are easily available and provide an equivalent or better yield of lumpy skin disease virus. Foetal bovine muscle (FBM) cells were found to be an adequate alternative for lamb testis cells.