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Liu F.,National Research Center for Exotic Animal Diseases | Wu X.,National Research Center for Exotic Animal Diseases | Liu W.,Qingdao Agricultural University | Li L.,National Research Center for Exotic Animal Diseases | Wang Z.,National Research Center for Exotic Animal Diseases
Veterinary Research Communications | Year: 2014

Peste des petits ruminants (PPR) is an acute or subacute, highly contagious viral disease of small ruminants, characterized by fever, oculonasal discharges, stomatitis, diarrhoea and pneumonia. This disease is included in the OIE (Office International des Epizooties) list of notifiable terrestrial animal diseases. PPR was first described in the early 1940s in Côte d′Ivoire, and at present, PPR is mainly circulating in Western and Central Africa, the Arabian Peninsula and Southern Asia. Peste des petits ruminants virus (PPRV), the etiological agent of PPR, is classified into the genus Morbillivirus in the family Paramyxoviridae, as its biological and physicochemical features are closely related to the other morbilliviruses. The first homologous PPR vaccine was developed by an artificially attenuated PPRV, named as Nigeria 75/1, which has been widely used in the production of live attenuated vaccines to protect small ruminants. A new generation of PPR vaccine candidates can be genetically modified to differentiate infected from vaccinated animals (DIVA), which nevertheless is difficult to achieve by conventional vaccines. In this review, we systematically discussed a broad range of vaccines against PPR, including commercially available vaccines and potential vaccine candidates, and further DIVA strategies for immunization with the new generation vaccines. © 2014, Springer Science+Business Media Dordrecht. Source


Liu F.,Jilin University | Liu F.,National Research Center for Exotic Animal Diseases | Wu X.,National Research Center for Exotic Animal Diseases | Li L.,National Research Center for Exotic Animal Diseases | And 2 more authors.
Protein Expression and Purification | Year: 2013

Peste des petits ruminants virus (PPRV), the etiological agent of peste des petits ruminants, is classified into the genus Morbillivirus in the family Paramyxoviridae. The PPRV matrix (M) gene is composed of 1483 base pairs, encoding a 335 amino acids M protein with a molecular weight of approximately 38 kD. We have demonstrated previously that the full-length M protein was expressed at an extremely low level or not even expressed in Escherichia coli BL21 (DE3). In this study, the M protein was split into two truncated forms to be successfully expressed in E. coli at a high level using the pET30a (+) vector, respectively, by analysis of SDS-PAGE, western blot and MALDI-TOF-MS. The optimization of culture conditions led us to perform the recombinant protein induction with 0.2 mM IPTG at 28 C for 12 h, whereby both proteins nevertheless were expressed in the insoluble form. Therefore, both His-tagged proteins were purified under the denaturing condition using a commercially available kit. Balb/c mice were immunized with the complex of purified proteins and then effectively produced polyclonal antibodies, which reached to a relatively high titer by the analysis of ELISA. The specificity of the prepared polyclonal antibodies was checked by western blot and immunofluorescence, revealing them with the desirable specificity against both non-denatured and denatured M proteins. © 2013 Elsevier Inc. All rights reserved. Source


Liu F.,National Research Center for Exotic Animal Diseases | Liu F.,Jilin University | Wu X.,National Research Center for Exotic Animal Diseases | Li L.,National Research Center for Exotic Animal Diseases | And 2 more authors.
Protein Expression and Purification | Year: 2013

The baculovirus expression system (BES) has been one of the versatile platforms for the production of recombinant proteins requiring multiple post-translational modifications, such as folding, oligomerization, phosphorylation, glycosylation, acylation, disulfide bond formation and proteolytic cleavage. Advances in recombinant DNA technology have facilitated application of the BES, and made it possible to express multiple proteins simultaneously in a single infection and to produce multimeric proteins sharing functional similarity with their natural analogs. Therefore, the BES has been used for the production of recombinant proteins and the construction of virus-like particles (VLPs), as well as for the development of subunit vaccines, including VLP-based vaccines. The VLP, which consists of one or more structural proteins but no viral genome, resembles the authentic virion but cannot replicate in cells. The high-quality recombinant protein expression and post-translational modifications obtained with the BES, along with its capacity to produce multiple proteins, imply that it is ideally suited to VLP production. In this article, we critically review the pros and cons of using the BES as a platform to produce both enveloped and non-enveloped VLPs. © 2013 Elsevier Ltd. All rights reserved. Source


Liu F.,Jilin University | Liu F.,National Research Center for Exotic Animal Diseases | Wu X.,National Research Center for Exotic Animal Diseases | Li L.,National Research Center for Exotic Animal Diseases | And 3 more authors.
Comparative Immunology, Microbiology and Infectious Diseases | Year: 2013

In general, it is difficult to differentiate infected from vaccinated animals through vaccination with conventional vaccines, thereby impeding the serological surveillance of animal diseases. DIVA (differentiating infected from vaccinated animals) vaccine, originally known as marker vaccine, usually based on the absence of at least one immunogenic protein in the vaccine strain, allows DIVA in conjunction with a diagnostic test that detects antibodies against the antigens lacking in the vaccine strain. Virus-like particles (VLPs), composed of one or more structural proteins but no genomes of native viruses, mimic the organization and conformation of authentic virions but have no ability to self-replicate in cells, potentially yielding safer vaccine candidates. Since VLPs containing either monovalent or multivalent antigen can be produced in compliance with the requirements for serological surveillance, the use of VLP-based vaccines plays a promising role in DIVA vaccination strategies against animal diseases. Here, we critically reviewed VLPs and companion diagnostics with properties of DIVA for veterinary vaccine design, and three different VLPs as promising platforms for DIVA vaccination strategies in animals. © 2013 Elsevier Ltd. Source


Liu F.,National Research Center for Exotic Animal Diseases | Wu X.,National Research Center for Exotic Animal Diseases | Zhao Y.,National Research Center for Exotic Animal Diseases | Li L.,National Research Center for Exotic Animal Diseases | Wang Z.,National Research Center for Exotic Animal Diseases
Journal of Virological Methods | Year: 2014

Peste des petits ruminants virus (PPRV), an etiological agent of peste des petits ruminants (PPR), is classified into the genus Morbillivirus in the family Paramyxovirida. In this study, two full-length open reading frames (ORF) corresponding to the PPRV matrix (M) and haemagglutinin (H) genes underwent a codon-optimization based on insect cells, respectively. Two codon-optimized ORFs along with one native nucleocapsid (N) ORF were used to construct recombinant baculoviruses co-expressing the PPRV M, H and N proteins in insect cells. Analysis of Western blot, immunofluorescence, confocal microscopy and flow cytometry demonstrated co-expression of the three proteins but at different levels in insect cells, and PPR virus-like particles (VLPs) budded further from cell membrane based on self-assembly of the three proteins by viewing of ultrathin section with a transmission electron microscope (TEM). Subsequently, a small number of VLPs were purified by sucrose density gradient centrifugation for TEM viewing. The PPR VLPs, either purified by sucrose density gradient centrifugation or budding from insect cell membrane on ultrathin section, morphologically resembled authentic PPRVs but were smaller in diameter by the TEM examination. © 2014 Elsevier B.V. Source

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