Production of CCHF Virus-Like Particle by a Baculovirus-Insect Cell Expression System

Crimean-Congo Haemorrhagic Fever Virus (CCHFV) is a tick-born virus of the Nairovirus genus within the Bunyaviridae family,which is widespread and causes high fatality.The nucleocapsid of CCHFV is comprised of N proteins that are encoded by the S segment.In this research,the N protein of CCHFV was expressed in insect cells using a recombinant baculovirus.Under an electron microscope,Virus-Like Particles (VLPs) with various size and morphology were observed in cytoplasmic vesicles in the infected cells.Sucro...     

Serial expression of the truncated fragments of the nucleocapsid protein of CCHFV and identification of the epitope region

The Crimean-congo hemorrhagic fever virus (CCHFV) is a geographically widespread fatal pathogen. Identification of the epitope regions of the virus is important for the diagnosis and epidemiological studies of CCHFV infections. In this study, expression vectors carrying series truncated fragments of the NP (nucleocapsid protein) gene from the S fragment of CCHFV strain YL04057 were constructed. The recombinant proteins were expressed in E.coli and purified for detection. The antigenic of the truncated fragments of NP was detected with a polyclonal serum (rabbit) and 2 monoclonal (mAbs) (14B7 and 43E5) against CCHFV by Western-blot analyses. The results showed that the three expressed constructs, which all contained the region 235AA to 305AA could be detected by mAbs polyclonal serum. The results suggest that region 235-305 aa of NP is a highly antigenic region and is highly conserved in the NP protein.     

口蹄疫病毒样颗粒诱导表达型载体的构建及其BHK-21细胞池的筛获

瞬时表达是目前利用哺乳动物细胞表达口蹄疫病毒(foot-and-mouth disease virus, FMDV)衣壳蛋白的主流方法。为实现染色体稳定表达FMDV衣壳蛋白并高效组装出病毒样颗粒(virus like particles, VLPs)，本研究构建了piggyBac (PB)转座-组成型表达、PB转座-四环素(tetracycline, Tet)诱导型表达两套质粒。利用荧光蛋白标记技术，验证了质粒的功能。通过抗生素筛选得到了组成型表达P12A3C (WT/L127P)基因的BHK-21细胞池(C-WT、C-L127P)和诱导型表达P12A3C (WT/L127P)基因的BHK-21细胞池(I-WT、I-L127P)。荧光观察和PCR检测证明了绿色荧光蛋白、3C蛋白酶、反向四环素转录激活因子等基因的稳定整合。Western blotting、酶联免疫吸附法(enzyme linked immunosorbent assay, ELISA)实验证明了细胞池I-L127P具有更强的衣壳蛋白和VLPs生产能力。本研究首次实现了哺乳动物细胞染色体诱导表达FMDV衣壳蛋白，有助于推动哺乳动物生产FMDV VLPs疫苗的技术工艺，也为构建其他蛋白的哺乳动物细胞诱导型表达系统提供了参考。     

Generation of methylated violapyrones with improved anti-influenza A virus activity by heterologous expression of a type III PKS gene in a marine Streptomyces strain

Heterologous expression of the type III polyketide synthase (PKS) gene vioA in marine-derived Streptomyces youssoufiensis OUC6819 led to production of six violapyrones (VLPs), including four novel compounds VLPs Q–T (1–4) and two known compounds VLPs B and I (5 and 6). The structures of 1–4 were elucidated by a combination of spectroscopic analyses, including HR-ESIMS and 1D and 2D NMR data, demonstrating that 1–4 are novel VLPs which are methylated at 4-OH with their corresponding non-methylated counterparts to be VLP A, 5 and 6 and VLP C, respectively. Anti-influenza A [H1N1 (A/Virginia/ATCC1/2009) and H3N2 (A/Aichi/2/1968)] virus activity of compounds 1–6 as well as VLPs A and C were then evaluated using ribavirin as a positive control (IC₅₀?=?66.7 and 99.6?μM). The results revealed that these VLPs showed considerable anti-H1N1 and anti-H3N2 activities with IC₅₀ values of 30.6–132.4?μM and 45.3–150.0?μM, respectively. Notably, all the methylated VLPs displayed better anti-virus activity than their non-methylated counterparts, among which compound 3 (VLP S) exhibited the best activities. Interestingly, methylation at 4-OH has negative effect on the anti-MRSA (methicillin-resistant Staphylococcus aureus) activity instead, with methylated VLPs displaying decreased (2) or abolished (3 and 4) activities in comparison with each of their non-methylated counterparts.     

Delivery of chimeric hepatitis B core particles into liver cells

Aims: To display a liver‐specific ligand on the hepatitis B virus core particles for cell‐targeting delivery. Methods and Results: A liver cell–binding ligand (preS1) was fused at the N‐terminal end of the hepatitis B core antigen (HBcAg), but the fusion protein (preS1His₆HBcAg) was insoluble in Escherichia coli and did not form virus‐like particles (VLPs). A method to display the preS1 on the HBcAg particle was established by incorporating an appropriate molar ratio of the truncated HBcAg (tHBcAg) to the preS1His₆HBcAg. Gold immunomicroscopy showed that the subunit mixture reassembled into icosahedral particles, displaying the preS1 ligand on the surface of VLPs. Fluorescence microscopy revealed that the preS1 ligand delivered the fluorescein‐labelled VLPs into the HepG2 cells efficiently. Conclusions: Chimeric VLPs containing the insoluble preS1His₆HBcAg and highly soluble tHBcAg were produced by a novel incorporation method. The preS1 ligand was exposed on the surface of the VLPs and was shown to deliver fluorescein molecules into liver cells. Significance and Impact of Study: The newly established incorporation method can be used in the development of chimeric VLPs that could serve as potential nanovehicles to target various cells specifically by substituting the preS1 ligand with different cell‐specific ligands.     

A new strain of Crimean-Congo hemorrhagic fever virus isolated from Xinjiang,China

Crimean-Congo hemorrhagic fever virus (CCHFV) is a highly pathogenic tick-borne virus with a fatality rate of up to 50% in humans. CCHFV is widely distributed in countries around the world. Outbreaks of CCHFV infection in humans have occurred in prior years in Xinjiang Province, China. Epidemiological surveys have detected CCHFV RNA in ticks and animals; however, few isolates were identified. In this study, we identified and isolated a new CCHFV strain from Hyalomma asiaticum asiaticum ticks collected from north of Tarim Basin in Xinjiang, China. A preliminary investigation of infection and antigens expression of CCHFV was performed in newborn mice. The target tissues for CCHFV replication in newborn mice were identified. The analysis of the phylogenetic relationships with other Chinese strains suggested that diverse genotypes of CCHFV have circulated in Xinjiang for years. These findings provide important insights into our understanding of CCHFV infection and evolution as well as disease prevention and control for local residents.

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Hyalomma ticks on northward migrating birds in southern Spain: Implications for the risk of entry of Crimean‐Congo haemorrhagic fever virus to Great Britain

Crimean‐Congo haemorrhagic fever virus (CCHFV) is a zoonotic virus transmitted by Hyalomma ticks, the immature stages of which may be carried by migratory birds. In this study, a total of 12 Hyalomma ticks were recovered from five of 228 migratory birds trapped in Spring, 2012 in southern Spain along the East Atlantic flyway. All collected ticks tested negative for CCHFV. While most birds had zero Hyalomma ticks, two individuals had four and five ticks each and the statistical distribution of Hyalomma tick counts per bird is over‐dispersed compared to the Poisson distribution, demonstrating the need for intensive sampling studies to avoid underestimating the total number of ticks. Rates of tick exchange on migratory birds during their northwards migration will affect the probability that a Hyalomma tick entering Great Britain is positive for CCHFV. Drawing on published data, evidence is presented that the latitude of a European country affects the probability of entry of Hyalomma ticks on wild birds. Further data on Hyalomma infestation rates and tick exchange rates are required along the East Atlantic flyway to further our understanding of the origin of Hyalomma ticks (i.e., Africa or southern Europe) and hence the probability of entry of CCHFV into GB.     

Impact of climate change on risk of incursion of Crimean-Congo haemorrhagic fever virus in livestock in Europe through migratory birds

Aims: To predict the risk of incursion of Crimean‐Congo haemorrhagic fever virus (CCHFV) in livestock in Europe introduced through immature Hyalomma marginatum ticks on migratory birds under current conditions and in the decade 2075–2084 under a climate‐change scenario. Methods and Results: A spatial risk map of Europe comprising 14 282 grid cells (25 × 25 km) was constructed using three data sources: (i) ranges and abundances of four species of bird which migrate from sub‐Saharan Africa to Europe each spring, namely Willow warbler (Phylloscopus trochilus), Northern wheatear (Oenanthe oenanthe), Tree pipit (Anthus trivialis) and Common quail (Coturnix coturnix); (ii) UK Met Office HadRM3 spring temperatures for prediction of moulting success of immature H. marginatum ticks and (iii) livestock densities. On average, the number of grid cells in Europe predicted to have at least one CCHFV incursion in livestock in spring was 1·04 per year for the decade 2005–2014 and 1·03 per year for the decade 2075–2084. In general with the assumed climate‐change scenario, the risk increased in northern Europe but decreased in central and southern Europe, although there is considerable local variation in the trends. Conclusions: The absolute risk of incursion of CCHFV in livestock through ticks introduced by four abundant species of migratory bird (totalling 120 million individual birds) is very low. Climate change has opposing effects, increasing the success of the moult of the nymphal ticks into adults but decreasing the projected abundance of birds by 34% in this model. Significance and Impact of the Study: For Europe, climate change is not predicted to increase the overall risk of incursion of CCHFV in livestock through infected ticks introduced by these four migratory bird species.     

Species distribution and detection of Crimean Congo Hemorrhagic Fever Virus (CCHFV) in field-collected ticks in Ankara Province, Central Anatolia, Turkey

Ticks may act as vectors for a number of infectious diseases including Crimean Congo Hemorrhagic Fever (CCHF). The causative agent is Crimean Congo Hemorrhagic Fever Virus (CCHFV), a member of Bunyaviridae, causing extensive ecchymosis, visceral bleeding and hepatic dysfunction with a high fatality rate in the affected individuals. CCHF was initially recognized in Turkey in 2002 and the current number of reported cases exceeds 4,400. This study was conducted to confirm the presence of tick species established as potential CCHFV vectors and investigate CCHFV activity in ticks at Ankara province, Turkey’s second most-densely populated province, where CCHF cases were demonstrated. A total of 1,196 adult ticks, collected from various animals and vegetation in 12 sites located in 5 counties of Ankara during April–July 2010 were identified to species level. Twenty-two tick pools from county K2 were also evaluated for the presence of CCHFV RNA via a one-step real-time RT-PCR assay and reactive results were further confirmed by an in house nested RT-PCR assay. Nine tick species were identified: Rhipicephalus bursa (44.9%), R. sanguineus (18.9%), R. turanicus (18.1%), Haemaphysalis parva (8.3%), Hyalomma marginatum marginatum (5.4%), H. aegyptium (1.4%), H. anatolicum excavatum (1.3%), Hae. punctata (0.3%) and Dermacentor marginatus (0.2%). A total of five tick pools (22.7%) were reactive in real-time and nested RT-PCR assays. The pools included R. bursa, H. m. marginatum and Hae. parva ticks, collected from mammal hosts from two villages in one county. This is the first documentation of CCHFV activity in ticks from Ankara province, which indicates requirement for detailed surveillance to predict high risk zones in the region.     

Probable mechanical transmission of a virus-like agent from rose rosette disease-infected multiflora rose to Nicotiana species

As part of efforts to identify the causal agent of the rose rosette disease (RRD) of multiflora rose (Rosa multiflora Thunb.), root tip extracts from both symptomatic and nonsymptomatic roses were used to mechanically inoculate leaves of Nicotiana glutinosa. Pale green spots were observed along the margins of the major leaf veins only on leaves inoculated with extracts prepared from symptomatic rose plants. Light microscopy revealed abnormal development of the palisade and spongy mesophyll cells in the symptomatic tissue, although no virus‐like particles (VLPs) were observed by electron microscopy. However, VLPs were observed in cells from tissue adjacent to the leaf veins and bordered by the pale green spots. Inoculation of N. benthamiana with extracts from symptomatic N. glutinosa initially did not result in visible symptoms on N. benthamiana inoculated leaves. However, approximately 4 wk post inoculation, splitting of leaf tissue across and along major leaf veins in expanding leaves occurred. In later stages of leaf expansion some leaves split in regions not associated with veins. Light microscopy of thick sections revealed separation between palisade cells and groups of small dead cells in the mesophyll tissue of expanding systemically infected leaf blades. Electron microscopy revealed crystalline arrays in the cytoplasm of mesophyll cells. No abnormal cellular changes were observed in plants inoculated with extracts prepared from nonsymptomatic rose plants.     

Virus‐like particle of Macrobrachium rosenbergii nodavirus produced in Spodoptera frugiperda (Sf9) cells is distinctive from that produced in Escherichia coli

Macrobrachium rosenbergii nodavirus (MrNV) is a virus native to giant freshwater prawn. Recombinant MrNV capsid protein has been produced in Escherichia coli, which self‐assembled into virus‐like particles (VLPs). However, this recombinant protein is unstable, degrading and forming heterogenous VLPs. In this study, MrNV capsid protein was produced in insect Spodoptera frugiperda (Sf9) cells through a baculovirus system. Dynamic light scattering (DLS) and transmission electron microscopy (TEM) revealed that the recombinant protein produced by the insect cells self‐assembled into highly stable, homogenous VLPs each of approximately 40 nm in diameter. Enzyme‐linked immunosorbent assay (ELISA) showed that the VLPs produced in Sf9 cells were highly antigenic and comparable to those produced in E. coli. In addition, the Sf9 produced VLPs were highly stable across a wide pH range (2–12). Interestingly, the Sf9 produced VLPs contained DNA of approximately 48 kilo base pairs and RNA molecules. This study is the first report on the production and characterization of MrNV VLPs produced in a eukaryotic system. The MrNV VLPs produced in Sf9 cells were about 10 nm bigger and had a uniform morphology compared with the VLPs produced in E. coli. The insect cell production system provides a good source of MrNV VLPs for structural and immunological studies as well as for host–pathogen interaction studies. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:549–557, 2017     

Influenza virus‐like particles produced by transient expression in Nicotiana benthamiana induce a protective immune response against a lethal viral challenge in mice

A strain‐specific vaccine represents the best possible response to the threat of an influenza pandemic. Rapid delivery of such a vaccine to the world's population before the peak of the first infection wave seems to be an unattainable goal with the current influenza vaccine manufacturing capacity. Plant‐based transient expression is one of the few production systems that can meet the anticipated surge requirement. To assess the capability of plant agroinfiltration to produce an influenza vaccine, we expressed haemagglutinin (HA) from strains A/Indonesia/5/05 (H5N1) and A/New Caledonia/20/99 (H1N1) by agroinfiltration of Nicotiana benthamiana plants. Size distribution analysis of protein content in infiltrated leaves revealed that HA was predominantly assembled into high‐molecular‐weight structures. H5‐containing structures were purified and examination by transmission electron microscopy confirmed virus‐like particle (VLP) assembly. High‐performance thin layer chromatography analysis of VLP lipid composition highlighted polar and neutral lipid contents comparable with those of purified plasma membranes from tobacco plants. Electron microscopy of VLP‐producing cells in N. benthamiana leaves confirmed that VLPs accumulated in apoplastic indentations of the plasma membrane. Finally, immunization of mice with two doses of as little as 0.1 µg of purified influenza H5‐VLPs triggered a strong immune response against the homologous virus, whereas two doses of 0.5 µg of H5‐VLPs conferred complete protection against a lethal challenge with the heterologous A/Vietnam/1194/04 (H5N1) strain. These results show, for the first time, that plants are capable of producing enveloped influenza VLPs budding from the plasma membrane; such VLPs represent very promising candidates for vaccination against influenza pandemic strains.     

牛病毒性腹泻病毒1型病毒样颗粒的制备及其对豚鼠的免疫原性分析

为了获得预防牛病毒性腹泻病毒1型(bovine viral diarrhea virus 1,BVDV-1)感染的病毒样颗粒,扩增C-Ems-E1-E2编码区段并克隆至pFastBacDaul载体,转化大肠杆菌DH10Bac感受态细胞与Bacmid重组获得Bacmid-BVDV-1,转染至Sf9细胞,获得重组杆状病毒B...     

The Non-structural Protein of Crimean-Congo Hemorrhagic Fever Virus Disrupts the Mitochondrial Membrane Potential and Induces Apoptosis

Viruses have developed distinct strategies to overcome the host defense system. Regulation of apoptosis in response to viral infection is important for virus survival and dissemination. Like other viruses, Crimean-Congo hemorrhagic fever virus (CCHFV) is known to regulate apoptosis. This study, for the first time, suggests that the non-structural protein NSs of CCHFV, a member of the genus Nairovirus, induces apoptosis. In this report, we demonstrated the expression of CCHFV NSs, which contains 150 amino acid residues, in CCHFV-infected cells. CCHFV NSs undergoes active degradation during infection. We further demonstrated that ectopic expression of CCHFV NSs induces apoptosis, as reflected by caspase-3/7 activity and cleaved poly(ADP-ribose) polymerase, in different cell lines that support CCHFV replication. Using specific inhibitors, we showed that CCHFV NSs induces apoptosis via both intrinsic and extrinsic pathways. The minimal active region of the CCHFV NSs protein was determined to be 93–140 amino acid residues. Using alanine scanning, we demonstrated that Leu-127 and Leu-135 are the key residues for NSs-induced apoptosis. Interestingly, CCHFV NSs co-localizes in mitochondria and also disrupts the mitochondrial membrane potential. We also demonstrated that Leu-127 and Leu-135 are important residues for disruption of the mitochondrial membrane potential by NSs. Therefore, these results indicate that the C terminus of CCHFV NSs triggers mitochondrial membrane permeabilization, leading to activation of caspases, which, ultimately, leads to apoptosis. Given that multiple factors contribute to apoptosis during CCHFV infection, further studies are needed to define the involvement of CCHFV NSs in regulating apoptosis in infected cells.     

NADC30-like猪繁殖与呼吸综合征病毒样颗粒的制备与鉴定

【背景】猪繁殖和呼吸综合征病毒(porcine reproductive and respiratory syndrome virus,PRRSV)可以造成怀孕母猪的繁殖障碍及仔猪的呼吸系统疾病,近年来,NADC30-like谱系PRRSV已成为国内的优势流行毒株。【目的】研制针对NADC30-like谱系PRRSV的病毒样颗粒(virus-like particle,VLP)疫苗。【方法】将PRRSV NADC30-like毒株编码GP5蛋白开放阅读框5(open reading frame 5,ORF5)、ORF6(编码M蛋白)分别连接至pFastBac^TMDual载体P₁₀和P_H启动子下游多克隆位点,获得穿梭质粒pFB-30-ORF5及pFB-30-ORF6,酶切鉴定后,将ORF6基因插入到穿梭质粒pFB-30-ORF5 P_H启动子下游,构建穿梭质粒pFB-30-ORF5-OPF6。将上述3种穿梭质粒分别转化大肠杆菌DH10Bac感受态细胞,通过蓝白斑筛选及PCR鉴定重组杆粒。再将获得的重组杆粒转染至SF9昆虫细胞,发现细胞病变后收获病毒液,继续盲传3代,在透射电镜下观察是否有病毒样颗粒。用第3代病毒液感染SF9细胞后,分别用GP5蛋白、His-tag、Flag-tag单克隆抗体作为一抗,通过免疫电镜、间接免疫荧光(indirect immunofluorescence assay,IFA)、Western blotting鉴定重组蛋白。【结果】成功构建了3种穿梭质粒pFB-30-ORF5、pFB-30-ORF6和pFB-30-ORF5-OPF6,酶切鉴定正确。通过蓝白斑筛选及PCR验证后获得重组杆粒,分别命名为Bacmind-30-ORF5、Bacmind-30-ORF6和Bacmind-30-ORF5-ORF6。重组杆粒感染SF9细胞120h时出现明显的细胞病变,收获病毒液后,在透射电子显微镜可观察到大小为50nm左右呈现球形结构的VLPs。免疫电镜可以观察到胶体金颗粒结合在VLPs周围;IFA结果显示实验组均出现了明显绿色的特异性荧光灶;Western blotting结果表明,3种VLPs均出现特异性条带,并与预期大小一致。【结论】制备了3种NADC30-like谱系PRRSV的病毒样颗粒,为针对PRRSV新谱系流行株疫苗的研发奠定了基础。     

Induction of protective immune response to intranasal administration of influenza virus-like particles in a mouse model

Human influenza A viruses (IAVs) cause global pandemics and epidemics, which remains a nonignorable serious concern for public health worldwide. To combat the surge of viral outbreaks, new treatments are urgently needed. Here, we design a new vaccine based on virus-like particles (VLPs) and show how intranasal administration of this vaccine triggers protective immunity, which can be exploited for the development of new therapies. H1N1 VLPs were produced in baculovirus vectors and were injected into BALB/c mice by the intramuscular (IM) or intranasal (IN) route. We found that there were significantly higher inflammatory cell and lymphocyte concentrations in bronchoalveolar lavage samples and the lungs of IN immunized mice; however, the IM group had little signs of inflammatory responses. On the basis of our results, immunization with H1N1 influenza VLP elicited a strong T cell immunity in BALB/c mice. Despite T cell immunity amplification after both IN and IM vaccination methods in mice, IN-induced T cell responses were significantly more intense than IM-induced responses, and this was likely related to an increased number of both CD11b^high and CD103⁺ dendritic cells in mice lungs after IN administration of VLP. Furthermore, evaluation of interleukin-4 and interferon gamma cytokines along with several chemokine receptors showed that VLP vaccination via IN and IM routes leads to a greater CD4⁺ Th1 and Th2 response, respectively. Our findings indicated that VLPs represent a potential strategy for the development of an effective influenza vaccine; however, employing relevant routes for vaccination can be another important part of the universal influenza vaccine puzzle.