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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.


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.


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.


PubMed | Yangzhou University and National Research Center for Exotic Animal Diseases
Type: Journal Article | Journal: Transboundary and emerging diseases | Year: 2016

Re-emergence of peste des petits ruminants (PPR) was officially reported in Xinjiang Uygur Autonomous Region in north-western China in November 2013, and then along with the movements of goats and sheep, this disease rapidly spread to other provinces, autonomous regions and municipalities (P/A/M) of China. A total of 256 PPR-affected counties in 22 P/A/M were identified up to September 2014. Phylogenetic analysis revealed that the current circulating strains and Tibet strains isolated previously in 2007, both belonged to lineage IV but in different sub-branches. Nevertheless, compared with the Tibet strains, the current circulating strains shared high degree of genetic homology with those from Pakistan and Tajikistan.


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.


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.


Liu F.,National Research Center for Exotic Animal Diseases | Wu X.,National Research Center for Exotic Animal Diseases | Zou 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: 2015

Peste des petits ruminants virus (PPRV), an etiological agent of peste des petits ruminants (PPR), is classified into the genus Morbillivirus in the family Paramyxoviridae. In a previous study, a recombinant baculovirus has been constructed to co-express the PPRV matrix (M), haemagglutinin (H) and nucleocapsid (N) proteins in insect cells, causing budding of PPR virus-like particles (VLPs) from insect cell membranes by viewing of ultrathin section with a transmission electron microscope. In this follow-up study, these PPR VLPs were purified by sucrose density gradient centrifugation for immunizing mice twice. Three weeks post-primary immunization and 2 weeks post-secondary immunization, all serum samples were obtained and subsequently subjected to indirect ELISA detection on complete virus-specific antibodies. In addition, all serum samples, which were collected 2 weeks post-secondary immunization, were used for virus neutralization test on PPRV neutralizing antibodies. The results showed that the purified PPR VLPs induced both types of antibodies mentioned above in mice, indicating a given potential of VLP-based vaccine candidate against PPR. © 2014 Elsevier B.V.


PubMed | National Research Center for Exotic Animal Diseases
Type: | Journal: Journal of virological methods | Year: 2016

Peste des petits ruminants virus (PPRV) is the cause agent of peste des petitis ruminants (PPR). A novel lineage IV PPRV has reemerged in China in 2013 and 2014. Mass vaccination was implemented in most provinces in China. In order to detect lineage IV PPRV in clinical samples and to distinguish rapidly it from the other lineages PPRVs, a real-time RT-PCR assay was developed. This assay showed high sensitivity, specificity and efficiency in differentiating the lineage IV PPRV from others. The performance of this assay was evaluated by positive clinical samples of lineage IV viruses. This new real-time RT-PCR assay will facilitate epidemiological investigations and rapid differentiatial diagnosis in areas where lineage IV viruses are circulating.


PubMed | National Research Center for Exotic Animal Diseases
Type: Journal Article | Journal: Virus genes | Year: 2016

Peste des petits ruminants (PPR) is an highly contagious disease of small ruminants, and caused by peste des petits ruminants virus (PPRV), a member of the genus Morbillivirus in the family Paramyxoviridae. The first outbreak of PPR in China was officially reported in July 2007, when a PPRV strain was successfully isolated from a sick goat in Tibet, followed by sequencing at a full-genome level (China/Tibet/Geg/07-30, GenBank: FJ905304.1). To date, this isolate has been virulently attenuated by more than 90 serial passages in Vero-Dog-SLAM cells at our laboratory. In this study, a total of nine strains by serial passages (namely the 10th, 20th, 30th, 40th, 50th, 60th, 70th, 80th, and 90th passages) were chosen for sequencing of six structural genes in PPRV. The sequence analysis showed that mutation rates in all viral genes were relatively low, and only a few identical mutations within certain genes were stably maintained after an earlier passage, perhaps indicating a predominance of mutants after such a passage.


PubMed | National Research Center for Exotic Animal Diseases
Type: | Journal: Comparative immunology, microbiology and infectious diseases | Year: 2016

The genus Morbillivirus is classified into the family Paramyxoviridae, and is composed of 6 members, namely measles virus (MV), rinderpest virus (RPV), peste-des-petits-ruminants virus (PPRV), canine distemper virus (CDV), phocine distemper virus (PDV) and cetacean morbillivirus (CeMV). The MV, RPV, PPRV and CDV have been successfully attenuated through their serial passages in vitro for the production of live vaccines. It has been demonstrated that the morbilliviral virulence in animals was progressively attenuated with their consecutive passages in vitro. However, only a few reports were involved in explanation of an attenuation-related mechanism on them until many years after the establishment of a quasispecies theory. RNA virus quasispecies arise from rapid evolution of viruses with high mutation rate during genomic replication, and play an important role in gradual loss of viral virulence by serial passages. Here, we overviewed the development of live-attenuated vaccine strains against morbilliviruses by consecutive passages in vitro, and further discussed a related mechanism concerning the relationship between virulence attenuation and viral evolution.

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