Function of PRRSV GP5 envelope protein by using pseudotyped virus.

The porcine reproductive and respiratory syndrome virus (PRRSV) is an enveloped RNA virus. Virions of PRRSV contain six membrane proteins: the major proteins GP5 and M and the minor proteins GP2, GP3, GP4, and E. The GP5 is the major envelope proteins, which was involved in the formation and infectivity of PRRSV by coaction with other membrane proteins. Here, to determine the function of alone GP5 envelope protein in viral entry, we investigated the formation and infectivity of GP5-pseudotyped virus particles. By co-transfection of GP5 expression plasmids with murine leukemia virus (MuLV) based retroviral vectors (pHIT60, encoding MuLV Gag-Pol; pHIT111, encoding an MuLV genome with a beta-galactosidase reporter gene) into 293 T cells and analysis of the culture medium using ultracentrifugation, Western blot, and infection assay. We observed that the GP5 envelope protein was incorporated into the MuLV retroviral vectors to generate an pseudotyped murine leukemia virus, which was infectious to PAM and Mack-145 target cells and displayed the same host range with wild-type PRRSV. The infection of the pseudotyped virus on PAM target cells is effectively neutralized by polyclonal antibodies specific for PRRSV or GP5. The results suggested that the GP5 protein may play a key role in the viral entry by interacting with the host cell receptor. The GP5-pseudotyped virus will be useful in the identification of the cellular receptor binding with GP5 protein.

[Pseudotyping of murine leukemia virus particles with porcine reproductive and respiratory syndrome virus M protein-mediated E protein].

For constructing pcDNA-ORF5, pcDNA-ORF5-ORF6, pcDNA-ORF5/6, the ORF5 and ORF6 of porcine reproductive and respiratory syndrome virus (PRRSV) were amplified by RT-PCR, and transiently transfected into 293T cells by calciumphosphate co-precipitation. After 48 h, 293T cells were collected and surveyed by flow cytometry examination. The result indicated that the expression level of the E protein that mediated by the M protein was higher than that of the E protein expressed independently. Then pcDNA-ORF5, pcDNA-ORF5-ORF6, pcDNA-ORF5/6 were respectivly co-transfected into 293T cells with pHIT60 (include MuLV structural genes,namely gag and pol) and pHIT111 (retroviral genome, containing LacZ as a reporter). The supernatants were harvested 48 h post-transfection,and the analysis of the characteristic of the pseudotyping virions was performed by Western blot and infection test. The result indicated that the E proteins were expressed on the virions, and incorporated into the retroviral virions. Infection test were performed on Marc-145 and PAM, all the cells infected were Lac Z positive. These results indicated the pseudotype virions of MuLV-E and MuLV-E/M were infectious, and higher infectivity was achieved by MuLV-E/M.

Bursin as an adjuvant is a potent enhancer of immune response in mice immunized with the JEV subunit vaccine.

Adjuvants play an important role in the formulation of effective and appropriate vaccines. A series of synthetic bursin and bursin-like peptides was heterologously expressed in Escherichia coli. The use of bursin as an adjuvant enhanced the specific immune responses of mice immunized with a recombinant Japanese encephalitis virus E-binding domain (JEV E-BD) fusion protein. The effect was determined in the form of protective anti-JEV E titers, antibodies (IgG1 and IgG2a), spleen cell lymphocyte proliferation, the levels of interferon-gamma and interleukin-4 cytokines, and the T-lymphocyte sub-type composition. The IgG2a titer and interferon-gamma level suggested that the E-BD protein potentiates the Th1 immune response. These responses were changed when the immunogen was combined with one of the synthetic peptides as adjuvant. JEV-neutralization assay results show that the presence of bursin significantly enhance the JEV-neutralizing titer. We conclude that bursin as an adjuvant is a potent enhancer of immune response in mice immunized with the JEV subunit vaccine, and represents a promising adjuvant for vaccination.