Fig 1.
Strategies for constructing the MDV-1 Md5 BAC clone.
(A) Schematic of the linear MDV-1 Md5 genome and a portion of the unique short region (US) containing US10 to US6 ORFs and the location of homologous arms (horizontal dashed line). (B) A linearized transfer vector, pBlue-US2-BAC-RFP, includes homologous arms (US2A and US2B), a loxP-flanked partial sequence of the pBeloBAC11 plasmid, and a selection marker expression cassette (RFP and puromycin). (C) CEFs were transfected with linearized pBlue-US2-BAC-RFP, followed by MDV-1 Md5 virus infection. Through classical homologous recombination, the transfer vector was introduced into the MDV-1 genome between SORF3 and US3 to yield a recombinant MDV-1, Md5-BAC-RFP. (D) Microscopic observation of red fluorescent plaques of Md5-BAC-RFP obtained by plaque purification in CEFs. (E) Md5-BAC-RFP viral DNA was isolated from infected CEFs and electroporated into E. coli DH10B cells to generate infectious pMd5 clones. (F and G) CEFs were transfected with pMd5 DNA with or without the Cre recombinase expression plasmid and produced recombinant virus rMd5-BAC-RFP (F) or rMd5 (G). The blue arrows indicate the locations of primers used to confirm the removal of the BAC sequence.
Fig 2.
Characterization of the BAC-derived viruses in vitro and in vivo.
(A) RFLP analysis of BAC DNA. Electrophoresis of pMd5 DNAs (clones 14–4 and 14–20) that were cleaved with EcoRI, BamHI, HindIII, or SacI endonucleases. The bands that indicate the insertion of the transfer vector were marked with asterisks. (B) The microscopic images of viral plaques of Md5, rMd5/14-4, and rMd5/14-20 in CEFs at 5 dpi. (C) The growth curve of Md5, rMd5/14-4, or rMd5/14-20 was performed as described in “Materials and Methods.” Each value represents the mean of two independent duplicates. (D-G) One-day-old SPF chickens were randomly assigned to receive intraperitoneal injections of either Md5 or rMd5 at a dose of 1,000 PFU, or DMEM as a control. The chickens were kept in isolators for 54 days together with contact chickens housed in the same isolators as either the Md5 or rMd5 group. (D) Survival curves of chickens inoculated with the indicated viruses and the control group. (E) The viral load in contacted chickens. The levels of the MDV VP22 and ICP4 genes were measured by PCR using DNA purified from the spleen tissues of mock-treated or contact chickens from the Md5 or rMd5 groups at 54 dpi. (F and G) Liver (F) and heart (G) tissues from chickens at 42 dpi in each group were stained with H&E. The black boxes indicate large populations of lymphoid tumor cells scattered in the Md5- or rMd5-infected tissues. Scale bar: 50 μm.
Fig 3.
Construction of the pLORF1-deficient virus.
(A) Schematic diagrams of the genome structure of MDV Md5 and the mutant viruses. Line 1: the wild-type MDV Md5 genome; Line 2: a segment of the Md5 genome that encompasses the R-LORF12 to LORF2 ORFs; Line 3: the rMd5ΔLORF1 virus with newly created stop codons (asterisks) due to a 2-bp deletion in the LORF1 gene; Line 4: the rMd5-reLORF1 virus with restored wild-type LORF1 ORF by re-introduction of 2 bp in the mutant rMd5ΔLORF1. The C-terminal region of pLORF1 (174–333 aa) was expressed as an antigen to produce anti-pLORF1 antibodies (black arrow). (B) RFLP analysis of HindIII-digested BAC DNAs from pMd5, pMd5ΔLORF1, or pMd5-reLORF1. (C) PCR amplification and sequencing results of the LORF1 gene in pMd5ΔLORF1 or -reLORF1. (D) The subcellular localization of pLORF1. CEFs were infected with rMd5, rMd5ΔLORF1, or rMd5-reLORF1 for 3 days, followed by fixation and staining with anti-pLORF1 antibody and Alexa 555-conjugated goat anti-mouse IgG antibody (red). The nuclei were stained with DAPI (blue). Original magnification: ×600. (E) The growth curve of rMd5, rMd5ΔLORF1, or rMd5-reLORF1. Each value represents the mean of two independent duplicates.
Fig 4.
Loss of pLORF1 attenuates MDV-induced pathogenicity and bursal atrophy.
The animal experiment was performed as described in “Materials and Methods.” (A) Survival curves of chickens inoculated with the indicated viruses and the control group. The time interval indicated by the two red dotted lines represents when 82% (9/11 chickens) of rMd5ΔLORF1-infected chickens died. (B) The body weight of dead chickens in each group from 35 dpi to 60 dpi. (C) The bursa to body weight ratio of chickens from each group at the indicated time points. Results represent the mean value, with error bars indicating the standard error of the mean. (D) The level of the MDV VP22 gene was measured by PCR using DNA purified from bursa and spleen samples of chickens from each group at the indicated time points.
Fig 5.
Histopathological examination of bursal tissues infected by recombinant viruses.
The bursa tissues from chickens that were mock-treated or inoculated with 1,000 PFU of rMd5, rMd5ΔLORF1, or rMd5-reLORF1, and collected at 7 dpi (A-D) and 42 dpi (E-H). (A and E) Low-magnification images of HE-stained bursa tissues. (B and F) A higher magnification of the areas indicated by red boxes in A or E. (C and G) Low-magnification images of immunohistochemistry of bursa tissues. Bursal sections stained with anti-VP22 antibody and HRP-conjugated goat anti-mouse IgG antibody were visualized using DAB (brown), while the nuclei were stained with hematoxylin (blue). (D and H) Higher magnifications of the areas indicated by the red boxes in panels C or G. (I) Bursal sections at 7 dpi were stained with anti-Bu-1 and Alexa 488-conjugated goat anti-mouse IgG antibodies (green), followed by staining with anti-VP22 and Alexa 555-conjugated goat anti-mouse IgG antibodies (red). The nuclei were stained with DAPI (blue). CTS, Connective Tissue Septum; CME, Cortico-Medullary Epithelium; C, Cortex; M, Medulla. Scale bar: 1 mm (A, C, E, and G), 20 μm (B, D, F, and H).
Fig 6.
Analysis of the expression pattern of pLORF1.
(A) Schematic diagram of pLORF1. (B and C) Membrane flotation analysis was conducted as described in the “Materials and Methods.” (B) Image of a 72%-65%-10% sucrose gradient of extracts from Flag-pLORF1-transfected DEF cells after ultracentrifugation. The mass of floated cellular membranes is marked by a black arrow. (C) Gradient fractions were analyzed using Western blotting with anti-Flag or anti-GFP antibodies. Fractions 1–5 correspond to the 65%-10% interface, which contains cellular membranes, while fractions 10–12 contain non-membrane-associated proteins. (D) Cellular localization of GFP-tagged pLORF1, VP22, VP11/12, pUL11, and pUL51. 293T cells were transfected with the indicated plasmids for 24 hours, then fixed and mounted in DAPI (blue). (E) Cellular localization of Flag-tagged pLORF1, VP22, VP11/12, pUL11, and pUL51. DF-1 cells were transfected with the indicated plasmids for 24 hours, followed by fixation and staining with anti-Flag antibody and Alexa 488-conjugated goat anti-mouse IgG antibody. (F) Cellular localization of pLORF1 in cells infected with rMDV. CEFs were infected with rMd5-GFP-pLORF1 for 3 days, fixed, mounted in DAPI, and analyzed by confocal microscopy. (G) Cellular localization of HA-tagged pLORF1 in cells infected with rMDV. CEFs were infected with rMd5-HA-pLORF1 for 3 days, fixed, and stained with anti-HA antibody and Alexa 488-conjugated goat anti-rabbit IgG antibody (green), followed by staining with anti-pLORF1 antibody and Alexa 555-conjugated goat anti-mouse IgG antibody (red). Scale bar: 10 μm (D and G); 20 μm (E and F).
Fig 7.
Analysis of interactions of pLORF1 with MDV structural proteins.
(A-C) DF-1 cells (A) were co-transfected with Flag-tagged pLORF1 and HA-tagged MDV-1 proteins (VP22, VP11/12, VP13/14, or VP19C) for 36 hours. 293T cells (B) were co-transfected with Flag-tagged pLORF1 and HA-tagged HSV-1 proteins (VP22, VP11/12, VP13/14, or VP19C) or EBV BGLF2 for 36 hours. CEFs (C) were transfected with Flag-tagged pLORF1 for 6 hours and subsequently infected with rMd5-HA-VP22, rMd5-HA-VP11/12, rMd5-HA-VP13/14, or rMd5-HA-VP19C for 2 days. Co-immunoprecipitation assays were performed using whole cell lysates with anti-Flag antibody or control IgG. Immunocomplexes were analyzed by immunoblotting with anti-Flag and anti-HA antibodies. (D) Colocalization of pLORF1 with rMDV structural proteins. CEFs were infected with rMd5-HA-pLORF1, rMd5-HA-VP22, rMd5-HA-VP11/12, rMd5-HA-VP13/14, or rMd5-HA-VP19C for 3 days, then fixed and stained with anti-HA antibody and either Alexa 488 or Alexa 555-conjugated goat anti-rabbit IgG, followed by staining with anti-pLORF1 antibody and either Alexa 555 or Alexa 488-conjugated goat anti-mouse IgG.
Table 1.
Primers used in this study.