Response of white-tailed deer to infection with peste des petits ruminants virus

White-tailed deer (Odocoileus virginianus) were infected experimentally with two strains of peste des petits ruminants virus. The response varied from fatal consequence to subclinical infection. The clinical signs and gross lesions were similar to those in goats. Virus was recovered from all the infected deer, and survivors developed specific antibodies demonstrated by complement fixation and virus neutralization tests. Survivors also resisted challenge with virulent rinderpest virus that was lethal to a control deer.     

西藏株小反刍兽疫病毒H蛋白的原核表达及其多克隆抗体的制备

【背景】小反刍兽疫是由小反刍兽疫病毒(Peste des petits ruminants virus,PPRV)引起的一种急性、烈性、接触性传染病,严重威胁我国养羊业的发展。【目的】原核表达PPRVH蛋白,并制备其多克隆抗体。【方法】根据GenBank中PPRV西藏株h基因序列,对其进行密码子大肠杆菌偏爱性优化,采用两步PCR法全化学合成全长h基因。将测序验证正确的h基因克隆至原核表达载体pET-28a、pET-30a、pET-32a,转化E. coli BL21(DE3)并利用IPTG诱导H蛋白表达。以经SDS-PAGE割胶纯化的重组H蛋白免疫新西兰大白兔制备抗PPRV H蛋白多克隆抗体。【结果】重组E. coli [pET-28a(-30a,-32a)-H]表达的重组H蛋白相对分子质量分别约为70、68和86 kD;诱导7 h时PRRV H蛋白表达量最高,而且主要以包涵体形式表达;重组E.coli(pET-30a-H)表达的H蛋白经SDS-PAGE割胶纯化后免疫新西兰大白兔制备的多抗血清能与表达的重组H蛋白发生特异性反应;ELISA法检测抗体效价在1:6400-1:25600之间。【结论】原核表达了PPRVH蛋白,并制备了高效价的抗H蛋白多克隆抗体,为进一步研究PPRV H蛋白的功能及H蛋白的线性B细胞表位作图奠定了基础。     

Comparative efficacy of peste des petits ruminants (PPR) vaccines

Peste des petits ruminants (PPR) is a highly contagious, economically important viral disease of sheep and goats with high morbidity and mortality rates. In order to control the disease effectively, highly sensitive diagnostic tests coupled with potent vaccines are important pre-requisites. At present, there are three live attenuated PPR vaccines available in India including Sungri 96, Arasur 87 and Coimbatore 97. Indian Veterinary Research Institute (IVRI) Mukteswar developed the PPR Sungri 96 (isolate of goat origin) vaccine; while Tamil Nadu Veterinary and Animal Sciences University (TANUVAS) developed the Arasur 87 (isolate of sheep origin) and Coimbatore 97 (isolate of goat origin). In this study, the potency of these vaccines including a fourth vaccine from Institute of Animal Health and Veterinary Biologicals, Bangalore (IAH&VB) were tested as per the office International des Epizooties (OIE) guidelines by challenge studies in sheep and goats and their efficacies were evaluated using PPR C-ELISA. Potency tests of these vaccines in sheep and goats revealed that three of the vaccines were potent; however, the IAH &VB vaccine was comparatively less potent. The three vaccines could presumably be used for mass vaccination of both sheep and goats while contemplating PPR control program.     

Enzymatic reporting of peste des petits ruminants virus genes ligating two specific probes on nanoparticles

An alternative strategy for the detection of nucleic acid derived from peste des petits ruminants virus was developed omitting amplification. The assay is based on two probes complementary to the target sequences, one conjugated to magnetic microparticles the second to gold nanoparticles labeled with horseradish peroxidase. In the presence of target gene the two particles ligate via the probes and the complex can be magnetically separated. Applying substrate and chromogen a color reaction results for a positive case. Under optimized conditions, the approach had a linear detection range from 10 fM to 1 μM for ssDNA corresponding to an RNA low detection limit of 17.6 ng/μl. The quick performance (45 min) and not requiring expensive instrumentations offer a new way of detecting nucleic acids for the clinical diagnosis in our case for peste des petits ruminants virus.     

Production and characterisation of monoclonal antibodies to an Indian isolate of peste des petits ruminants virus

Nine monoclonal antibodies (Mabs) were produced against an Indian isolate of peste des petits ruminants (PPR) virus. These Mabs were directed against the nucleo (N) protein and were of IgG1 isotype. The Mabs produced intranuclear or coarse granular cytoplasmic fluorescence in PPR virus infected Vero cells and did not exhibit any neutralising activity. The Mabs cross-reacted with five other local isolates of PPR virus in slot blot hybridisation, radio immunoprecipitation assay (RIPA) and fixed-cell enzyme linked immunosorbent assay (ELISA). Two of the nine Mabs cross-reacted mildly with the vaccine strain of rinderpest (RP) virus in slot blot hybridisation and fixed-cell ELISA but did not precipitate the N protein of RP virus in RIPA. The N protein specific Mabs will be highly useful in differential diagnosis of PPR from RP.     

Complete genome sequence of a peste des petits ruminants virus recovered from wild bharal in tibet, china

For the first time, here we announce the complete genome sequence of a field isolate of Peste des petits ruminants virus (PPRV) derived from macerated rectal tissue of a free living bharal (Pseudois nayaur) that displayed clinical disease consistent with severe infection with PPRV. Further, we compare the full genome of this isolate, termed PPRV Tibet/Bharal/2008, with previously available PPRV genomes, including those of virus isolates from domestic small ruminants local to the area where the reported isolate was collected. The current sequence is phylogenetically classified as a lineage IV virus, sharing high levels of sequence identity with previously described Tibetan PPRV isolates. Indeed, across the entire genome, only 26 nucleotide differences (0.16% nucleotide variation) and, consequently, 9 amino acid changes were present compared to sequences of locally derived viruses.     

The effects of codon usage on the formation of secondary structures of nucleocapsid protein of peste des petits ruminants virus

The nucleocapsid (N) protein of peste des petits ruminants virus (PPRV) with a conserved amino acid usage pattern plays an important role in viral replication. The primary objective of this study was to estimate roles of synonymous codon usages of PPRV N gene and tRNA abundances of host in the formation of secondary structure of N protein. The potential effects of synonymous codon usages of N gene and tRNA abundances of host on shaping different folding units (α-helix, β-strand and the coil) in N protein were estimated, based on the information about the modeling secondary structure of PPRV N protein. The synonymous codon usage bias was found in different folding units in PPRV N protein. To better understand the role of translation speed caused by variant tRNA abundances in shaping the specific folding unit in N protein, we modeled the changing trends of tRNA abundance at the transition boundaries from one folding unit to another folding unit (β-strand → coil, coil → β-strand, α-helix → coil, coil → α-helix). The obvious fluctuations of tRNA abundance were identified at the two transition boundaries (β-strand → coil and coil → β-strand) in PPRV N protein. Our findings suggested that viral synonymous codon usage bias and cellular tRNA abundance variation might have potential effects on the formation of secondary structure of PPRV N protein.     

从消灭牛瘟分析全球根除小反刍兽疫的可行性

根除,又称为消灭,是指某传染病的传播永远停止,不再发生。2011年,联合国粮农组织和国际兽疫局联合发布了全球根除牛瘟的消息。这两大国际组织的官方信息预示着,牛瘟病毒,正如天花病毒,将来只能在授权的实验室中保存了。牛瘟根除之后,相关学者不禁把目光投向了下一个目标-小反刍兽疫,因为从病原学和病理学等诸多方面考虑,二者最为相似。本文首先分析了前者根除的主客观因素,然后从正反两方面评述了后者全球性根除的可行性。     

SLAM (CD150) receptor homologous peptides block the peste des petits ruminants virus entry into B95a cells

The fusion of haemagglutinin-neuraminidase (HN) protein of peste des petits ruminant (PPR) virus with signaling lymphocyte activation molecules (SLAM) host cell receptor consequences the virus entry and multiplication inside the host cell. The use of synthetic SLAM homologous peptides (i.e., molecular decoy for HN protein of PPR virus) may check PPR infection at the preliminary stage. Hence, the predicted SLAM homologous peptides using bioinformatics tools were synthesized by solid phase chemistry with standard Merrifield's 9-fluorenylmethoxycarbonyl (Fmoc) chemistry and were purified by reverse phase high performance liquid chromatography. The secondary structures of synthesized peptides were elucidated by circular dichroism spectroscopy. The in vitro interactions of these peptides were studied through indirect Enzyme Linked Immuno Sorbent Assay (ELISA) and visual surface plasmon UV-visible spectroscopy. The SLAM homologous peptides were able to interact with the peste des petits ruminant virus (PPRV) with varying binding efficiency. The interaction of SLAM homologous peptide with the PPR virus was ascertained by the change in the plasmon color from red wine to purple during visual detection and also by bathochromic shift in absorbance spectra under UV-visible spectrophotometry. The cytotoxic and anti-PPRV effect of these peptides were also evaluated in B95a cell line using PPR virus (Sungri/96). The cytotoxic concentration 50 (CC₅₀) value of each peptide was greater than 1000 μg mL⁻¹. The anti-PPRV efficiency of SLAM-22 was relatively high among SLAM homologous peptides, SLAM-22 at 25 μg mL⁻¹ concentration showed a reduction of more than log₁₀ 3 virus titer by priming of B95a cell line while the use of SLAM-15 and Muco-17 at the same concentration dropped virus titer from log₁₀ 4.8 to log₁₀ 2.5 and log₁₀ 3.1 respectively. The concentration of SLAM homologous peptide (25 μg mL⁻¹) to exert its anti-PPRV effect was much less than its CC₅₀ level (>1000 μg mL⁻¹). Therefore, the synthetic SLAM homologous peptides may prove to be better agents to target PPRV.     

Fluid, electrolyte and haematological changes in experimental peste des petits ruminants virus infection in West African dwarf goats

The changes in plasma volume, blood volume, erythrocyte indices, leucocyte indices and serum biochemical values during experimental pestes des petits ruminants (PPR) virus infection of West African dwarf goats (Fouta djallon) were investigated. There was a progressive decline of the plasma volume and serum sodium and potassium values in all the infected goats as from day 5 post-infection and this became quite marked between days 10 and 23 post-infection. The fall was severest in those animals which had diarrhoea. There were no significant changes in the chloride, bicarbonate, phosphate, urea, total protein and albumin values of the goats during the course of the diseases. Haemoconcentration and apparent polycythaemia in the early and terminal stages of the disease were associated with severe dehydration which characterized the infection. There was initial neutrophilic leucocytosis during the phase of fever followed by marked lymphopaenic leucopaenia which progressed terminally in most of the infected goats. The destructive effect of the virus on lymphocytes is suggested as a cause of lymphopaenia.     

Six-helix bundle assembly and analysis of the central core of mumps virus fusion protein

The fusion protein of enveloped viruses mediates the fusion between the viral and cellular membranes, allowing the penetration of the viral genomes into the host cell. Many of these proteins share a common fold comprising a central core trimer of anti-parallel coiled-coil heterodimers, which are formed by two discontinuous heptad repeat (HR) motifs located at the ectodomain of the fusion proteins. In this study, we constructed and purified the corresponding regions (HR1 and HR2) of mumps virus fusion protein that are predicted to form coiled coil. The HR1 and HR2 were expressed and purified separately or as a single chain connected by an amino acid linker (HR1-linker-HR2, named 2-Helix). Series of biochemical and biophysical analyses of the expressed proteins have shown that HR1 and HR2 of mumps virus fusion protein share the common features of other enveloped virus fusion proteins. CD spectral results show that HR1 forms an alpha-helical coil structure while HR2 exists as an unstructured monomer in PBS in nature. Mixtures of HR1 and HR2 could form a stable six-helix bundle, indicating the interaction of HR1 and HR2. The 2-Helix protein also shows characteristic properties of the 6-helix bundle. Therefore, mumps virus fusion protein has a common core architecture and its HR regions could be used as a drug target for virus fusion inhibitors.     

Confirmed diagnosis by RT-PCR and phylogenetic analysis of peste des petits ruminants viruses in Tibet,China

This paper reports the confirmed diagnosis by nested RT-PCR of PPR cases in Tibet, China in 2007, and results of phylogenetic analysis. Results showed that the 11 tested samples were PPRV positive by nested RT-PCR, of which 2 samples were genetically close to the X7443 strain (Nigeria 75/1) of lineage I, and 3 samples close to the strain AY560591 (Sungri96) of linage IV with 96.6%、 97.3%、 97.6% and 98% nucleotide sequence homogeneity respectively, based on partial sequencing of the F gene from 5 samples and complete sequencing of the N/M/F/H genes from one sample. This study suggested that there are at least 2 origins of PPRV in China.     

Examination of epithelial tissue cytokine response to natural peste des petits ruminants virus (PPRV) infection in sheep and goats by immunohistochemistry

In this study, we aimed to evaluate expression of IL-4, IL-10, TNF-α, IFN-γ and iNOS in lingual, buccal mucosa and lung epithelial tissue using immunoperoxidase technique and to compare with the tissues of control animals. The tissues used in the study were collected from 17 PPRV-affected and 5 healthy sheep and goats. In PPRV positive animals, the lungs, lingual and buccal mucosa had significantly higher iNOS, IFN-γ and TNF-α expressions compared to control group animals. There was no significant difference between PPRV positive and control groups for IL-4 and IL-10 expressions of epithelial tissues. In conclusion, the epithelial tissues infected by PPRV showed significant iNOS, IFN-γ and TNF-α expressions and they might play an important role in the initiation and regulation of cytokine response, as they take place in the first host barrier to be in contact with PPRV. It is suggested that the more epithelial damage produced by PPRV the more cytokine response may result in the infected epithelial cells. The first demonstration of iNOS expression and epithelial cytokine response to PPRV in natural cases is important because it may contribute to an early initiation of systemic immunity against PPRV infection, in addition to direct elimination of the virus during the initial epithelial phase of the infection.     

Combining viral genetic and animal mobility network data to unravel peste des petits ruminants transmission dynamics in West Africa

Peste des petits ruminants (PPR) is a deadly viral disease that mainly affects small domestic ruminants. This disease threaten global food security and rural economy but its control is complicated notably because of extensive, poorly monitored animal movements in infected regions. Here we combined the largest PPR virus genetic and animal mobility network data ever collected in a single region to improve our understanding of PPR endemic transmission dynamics in West African countries. Phylogenetic analyses identified the presence of multiple PPRV genetic clades that may be considered as part of different transmission networks evolving in parallel in West Africa. A strong correlation was found between virus genetic distance and network-related distances. Viruses sampled within the same mobility communities are significantly more likely to belong to the same genetic clade. These results provide evidence for the importance of animal mobility in PPR transmission in the region. Some nodes of the network were associated with PPRV sequences belonging to different clades, representing potential “hotspots” for PPR circulation. Our results suggest that combining genetic and mobility network data could help identifying sites that are key for virus entrance and spread in specific areas. Such information could enhance our capacity to develop locally adapted control and surveillance strategies, using among other risk factors, information on animal mobility.     

青海岩羊源小反刍兽疫病毒N、F基因的遗传进化分析

2021年1月19日,青海省海西州都兰县巴隆乡伊克高里村发生岩羊不明原因的死亡,表现为离群独处、卧地不起、体质虚弱、觅食困难、肛门周围黑色附着物等现象。通过临床症状、病理学解剖及实时荧光RT-PCR诊断,为小反刍兽疫病毒(PPRV)感染。采用RT-PCR技术从病死岩羊病料组织中扩增出了PPRV N、F基因的部分片段。采用MegAlig、NT1和MEGA6.0软件对岩羊PPRV株N、F基因序列进行了比对和分析,绘制系统进化树,结果显示：此次岩羊感染的PPRV株N、F基因片段与新疆株(China/Xinjiang/2015/16)序列片段的同源性分别为99.43%和99.73%。遗传进化分析,该病原属于基因Ⅳ系。在N、F基因核酸序列水平上,与国内新疆地区分离毒株亲缘关系最近,同在一个小的分支;与国外毒株相比,N基因与塞内加尔和尼日利亚分离毒株亲缘关系较远,F基因与国外分离毒株亲缘关系较远。综上所述,青海岩羊源PPRV株属于基因Ⅳ系,与当前我国流行的野毒株属于同一个谱系。     

小反刍兽疫研究概况

小反刍兽疫是严重危害山羊和绵羊等家畜的A类动物传染病,目前该病已经给我国养羊业造成了巨大的威胁。近年来,国内外学者对该病开展了许多研究工作,本文从病原学、流行病学、临床症状、发病机理、病理变化、诊断、防控措施等几个方面对该病研究概况进行了综述,为该病的有效防控提供有益的参考。     

小反刍兽疫研究概况

小反刍兽疫是严重危害山羊和绵羊等家畜的A类动物传染病,目前该病已经给我国养羊业造成了巨大的威胁。近年来,国内外学者对该病开展了许多研究工作,本文从病原学、流行病学、临床症状、发病机理、病理变化、诊断、防控措施等几个方面对该病研究概况进行了综述,为该病的有效防控提供有益的参考。     

The coronavirus spike protein is a class I virus fusion protein: structural and functional characterization of the fusion core complex

Coronavirus entry is mediated by the viral spike (S) glycoprotein. The 180-kDa oligomeric S protein of the murine coronavirus mouse hepatitis virus strain A59 is posttranslationally cleaved into an S1 receptor binding unit and an S2 membrane fusion unit. The latter is thought to contain an internal fusion peptide and has two 4,3 hydrophobic (heptad) repeat regions designated HR1 and HR2. HR2 is located close to the membrane anchor, and HR1 is some 170 amino acids (aa) upstream of it. Heptad repeat (HR) regions are found in fusion proteins of many different viruses and form an important characteristic of class I viral fusion proteins. We investigated the role of these regions in coronavirus membrane fusion. Peptides HR1 (96 aa) and HR2 (39 aa), corresponding to the HR1 and HR2 regions, were produced in Escherichia coli. When mixed together, the two peptides were found to assemble into an extremely stable oligomeric complex. Both on their own and within the complex, the peptides were highly alpha helical. Electron microscopic analysis of the complex revealed a rod-like structure approximately 14.5 nm in length. Limited proteolysis in combination with mass spectrometry indicated that HR1 and HR2 occur in the complex in an antiparallel fashion. In the native protein, such a conformation would bring the proposed fusion peptide, located in the N-terminal domain of HR1, and the transmembrane anchor into close proximity. Using biological assays, the HR2 peptide was shown to be a potent inhibitor of virus entry into the cell, as well as of cell-cell fusion. Both biochemical and functional data show that the coronavirus spike protein is a class I viral fusion protein.     

基于N蛋白单克隆抗体的小反刍兽疫病毒抗体检测胶体金试纸条的研制

本研究旨在建立一种简便、快捷、可直观检测小反刍兽疫病毒(peste des petits ruminants virus,PPRV)抗体的检测方法。将pET-32a-N重组质粒转化至大肠杆菌(Escherichia coli) Rosetta(DE3)感受态细胞中进行诱导表达,以纯化的PPRVN蛋白免疫8周龄BALB/c小鼠,取其脾细胞与SP2/0骨髓瘤细胞进行融合,间接酶联免疫吸附试验(enzyme-linked immunosorbent assays, ELISA)筛选及亚克隆,获得了抗PPRV N蛋白的单克隆抗体。将PPRV N蛋白分别作为金标抗原及检测线(T线)包被抗原、单克隆抗体作为质控线(C线)包被抗体,组装成检测PPRVN蛋白抗体的胶体金免疫层析试纸条。结果显示：成功获得1株能稳定分泌抗N蛋白抗体的杂交瘤细胞株,命名为1F1;间接ELISA检测1F1腹水效价为1:128000;亚类鉴定结果为IgG1,轻链为kappa链。Westernblotting结果显示,1F1能与PPRV N蛋白特异性结合;间接免疫荧光(indirect immunofluorescent ass...