Journal: Viruses

Article Title: Cross Strain Protection against Cytomegalovirus Reduces DISC Vaccine Efficacy against CMV in the Guinea Pig Model

doi: 10.3390/v14040760

Figure Lengend Snippet: Comparative immune responses to DISC vaccine (DISCI vs. DISCII) and GPCMV (TAMYC strain) neutralization on fibroblast and epithelial cells. ( A – C ) Immune response of DISCII animal sera was compared to that of previous historical DISCI pooled sera from animals vaccinated with identical protocol . ( A ) Anti-GPCMV ELISA titer; ( B ) anti-gB glycoprotein ELISA titer; ( C ) anti-glycoprotein complex ELISA titers (gH/gL, gM/gN, PC) from sera of DISCI (black) or DISCII (gray) vaccinated animals. Neutralizing antibody titers (NA 50 ) against TAMYC strain virus on GPL (fibroblast) or REPI (epithelial) cells of pooled sera from either DISCI animals ( D ) or DISCII-vaccinated animals ( E ). Mean ELISA and neutralization values are a result of assay triplicates with each sample run a minimum of three independent times. Statistical analysis was determined by unpaired Student’s t test; ** p < 0.005; ns = non-significant.

Article Snippet: Animals purchased from Charles River Laboratories were verified as seronegative for GPCMV by anti-GPCMV ELISA of sera collected by toenail clip bleed as previously described [ ].

Techniques: Neutralization, Enzyme-linked Immunosorbent Assay, Virus

Journal: Viruses

Article Title: Cross Strain Protection against Cytomegalovirus Reduces DISC Vaccine Efficacy against CMV in the Guinea Pig Model

doi: 10.3390/v14040760

Figure Lengend Snippet: Comparative heterologous GPCMV (TAMYC strain) dissemination in convalescent (22122 strain) or control seronegative animals. Animals were infected with GPCMV (22122 strain) by single injection to establish convalescent natural immunity (22122-X1) or 3 sequential injections to establish hyperimmune status (22122-HI) prior to challenge (1 × 10 5 pfu, SQ) with GPCMV (TAMYC strain). Convalescent animals were evaluated for anti-GPCMV ELISA titer and specific anti-glycoprotein ELISA titers. ( A ) Mean anti-GPCMV ELISA titer comparison of sera from animals in 22122-X1 group (purple) or 22122-HI group (blue). ( B ) Comparative mean anti-glycoprotein complex ELISA titers (gB, gH/gL, gM/gN, PC) from sera of animals in 22122-X1 (purple) or 22122-HI group (blue). Statistical analysis determined by unpaired Student’s t test; ** p < 0.005; ns = non-significant. ( C – H ) Comparative GPCMV (TAMYC strain) dissemination in convalescent animals: ( C , D ) group 1 (22122-X1); ( E , F ) group 2 hyperimmune (22122-HI); or ( G , H ) group 3 control seronegative animals. Animals ( n = 12/group) were injected with 1 × 10 5 pfu, SQ of GPCMV (TAMYC strain). On days 4, 8, 12 and 27 post infection (dpi), 3 animals from each group were evaluated for viral load in target organs (lung, liver and spleen), by real-time PCR of DNA extracted from tissue. Viral load plotted as viral genome copies/mg tissue. Salivary gland (sal gland) tissue was only evaluated at 27 dpi ( C , E , G ). Viremia detected at 4, 8, 12 and 27 dpi was plotted as genome copies/mL blood ( D , F , H ).

Article Snippet: Animals purchased from Charles River Laboratories were verified as seronegative for GPCMV by anti-GPCMV ELISA of sera collected by toenail clip bleed as previously described [ ].

Techniques: Control, Infection, Injection, Enzyme-linked Immunosorbent Assay, Comparison, Real-time Polymerase Chain Reaction

Journal: Viruses

Article Title: Cross Strain Protection against Cytomegalovirus Reduces DISC Vaccine Efficacy against CMV in the Guinea Pig Model

doi: 10.3390/v14040760

Figure Lengend Snippet: GPCMV hyperimmune convalescent sera antibody responses and virus neutralization on fibroblast and epithelial cells. ( A – C ) Comparative ELISAs of 22122-HI (blue) or TAMYC-HI (orange) sera. ( A ) Mean anti-GPCMV ELISA titers; ( B ) mean anti-gB glycoprotein ELISA titers; ( C ) mean anti-glycoprotein complex ELISA titers (gH/gL, gM/gN, PC) of sera from animals infected with GPCMV 22122 strain (22122-HI, blue) or TAMYC strain (TAMYC-HI, orange). ( D – G ) Comparative GPCMV neutralization (NA 50 ) on GPL (fibroblast) or REPI (epithelial) cells by hyperimmune sera. ( D ) 22122-HI sera (blue) neutralization (NA 50 ) of 22122 strain virus. ( E ) TAMYC-HI sera (orange) NA 50 of TAMYC strain virus. ( F ) 22122-HI sera (blue) neutralization (NA 50 ) of TAMYC strain virus. ( G ) TAMYC-HI sera (orange) NA 50 of 22122 strain virus. Mean ELISA and neutralization values are a result of assay triplicates with each sample run a minimum of three independent times. Statistical analysis determined by unpaired Student’s t test; * p < 0.05; ** p < 0.005; ns = non-significant.

Article Snippet: Animals purchased from Charles River Laboratories were verified as seronegative for GPCMV by anti-GPCMV ELISA of sera collected by toenail clip bleed as previously described [ ].

Techniques: Virus, Neutralization, Enzyme-linked Immunosorbent Assay, Infection

Journal: PLoS Pathogens

Article Title: A Homolog Pentameric Complex Dictates Viral Epithelial Tropism, Pathogenicity and Congenital Infection Rate in Guinea Pig Cytomegalovirus

doi: 10.1371/journal.ppat.1005755

Figure Lengend Snippet: (i) Layout of the GPCMV GP128-133 locus of genes. Annotated GPCMV genome (co-ordinates 195,713–199,263 nucleotides) which encodes GP128 - gp134 . Individual genes represented as blue arrows and direction indicates either sense (above line) or complementary strand (below line) coding. Direct homologs to HCMV pentamer complex genes are UL128 (GP129) , UL130 (GP131) , UL131 (GP133) . GP129 (3 exons) and GP131 (two exons) are spliced genes. P1 (196,707) and P2 (198,701) show the location of the PCR primer pair (P1/P2) used to amplify the GP128-GP133 locus genes. The top line shows amplification of the full length locus (2 kb) from SG GPCMV. The bottom line shows amplification of the deleted GP128-GP133 locus in PP ATCC lab adapted virus (0.4 kb). Solid line indicates conserved sequence between full length locus and dotted line indicates deleted sequence. Co-ordinates 196,925–198,573 nucleotides represent the specific deletion within the locus for PP ATCC virus. Specific annotated nucleotide sequence of the GP128-GP133 locus is shown in . (ii) A. Analysis of full length and truncated viruses and epithelial cell infection. Agarose gel electrophoresis of the P1/P2 PCR for the GP128-133 locus from GPCMV. Left, kb ladder (Invitrogen). Middle, (ATTC) PP ATCC GPCMV (0.4 kb). Right, (SG) SG GPCMV (2 kb). B, C and D. Immunofluorescence assay of SG GPCMV infected epithelial cells. B. GPCMV IE2 detected with primary rabbit anti-IE2/ secondary anti rabbit IgG-FITC. C. GPCMV infected guinea pig epithelial cell monolayer verified by cytokeratin marker staining with primary mouse anti-pancytokeratin/ secondary anti-mouse IgG-TRITC. D. Overlay of B and C with cell nuclei stained with DAPI. (iii) Comparative growth curve of SG GPCMV and PP ATCC GPCMV on epithelial cells and fibroblast cells. Cells were infected with either at a moi of 1 pfu/cell. Sample were taken at different days post infection and titrated in duplicate on GPL cells as previously described . Results plotted as virus titer against days post infection: A, growth on epithelial cells; B, growth on GPL cells. Diamond (blue), SG GPCMV. Square (red), PP ATCC.

Article Snippet: Custom antibodies to GPCMV gH, IE2 and GP131 were generated by Genescript to specific peptide sequences (rabbit polyclonal) or to purified recombinant ( E . coli ) protein (mouse monoclonal).

Techniques: Amplification, Virus, Sequencing, Infection, Agarose Gel Electrophoresis, Immunofluorescence, Marker, Staining

Journal: PLoS Pathogens

Article Title: A Homolog Pentameric Complex Dictates Viral Epithelial Tropism, Pathogenicity and Congenital Infection Rate in Guinea Pig Cytomegalovirus

doi: 10.1371/journal.ppat.1005755

Figure Lengend Snippet: (i) Individual expression of pentameric complex components in epithelial cells transduced with recombinant adenovirus encoding either gHGFP, gLmCherry, GP129myc, GP131HA or GP133FLAG analyzed by western blot using respective primary antibodies: gH (anti-GFP); gL (anti-mCherry); GP129 (anti-myc); GP131 (anti-HA); GP133 (anti FLAG). Bands visualized with appropriate secondary antibody-HRP conjugate as described in materials and methods . Lys indicates defective adenovirus transduced lane and MI indicates mock cell lysate. (ii) Immunoprecipitation of the pentameric complex from epithelial cells transduced with all five recombinant defective adenoviruses encoding individual components (10 TDU/virus/cell). Samples were analyzed as total cell lysate as above or processed for immunoprecipitation assay. Immunoprecipitation was carried out with GFP trap as previously described . Individual proteins were detected as described for (i). Western blot lanes: 1–3 (anti-mCherry); 4–5 (anti-GFP); 6–8 (antiFLAG); 9–10 (anti-myc); 11–12 (anti-HA). Lanes 1 and 6 mock infected cell lysate. Lanes 2, 4, 7, 9 and 11 (total cell lysate). Lanes 3, 5, 8, 10 and 12 (immunoprecipitation). (iii) Control PC immunoprecipitation assay with GFP substituting for gHGFP. Epithelial cells transduced with defective recombinant adenoviruses encoding GFP, gLmCherry GP129myc, GP131HA and GP133FLAG followed by GFP immunoprecipitation and western blot for proteins as previously described . Western blot lanes: 1–4 (anti-GFP); 5–7 (anti-mCherry); 8–11 (anti-myc); 12–14 (anti-HA); 15–17 (anti-FLAG). Lanes 1 and 8 mock infected cell lysate. Lanes 2, 5, 9, 12 and 15 (total cell lysate). Lanes 3, 6, 10, 13 and 16 (flow through). Lanes 4, 7, 11, 14 and 17 (immunoprecipitation).

Article Snippet: Custom antibodies to GPCMV gH, IE2 and GP131 were generated by Genescript to specific peptide sequences (rabbit polyclonal) or to purified recombinant ( E . coli ) protein (mouse monoclonal).

Techniques: Expressing, Transduction, Recombinant, Western Blot, Immunoprecipitation, Virus, Infection, Control

Journal: PLoS Pathogens

Article Title: A Homolog Pentameric Complex Dictates Viral Epithelial Tropism, Pathogenicity and Congenital Infection Rate in Guinea Pig Cytomegalovirus

doi: 10.1371/journal.ppat.1005755

Figure Lengend Snippet: C-terminal GP129 mutants were evaluated for an ability to form pentamer complexes with gHGFP, gLmCherry, GP131HA and GP133FLAG. GP129 mutants were expressed on transient expression plasmids transfected onto epithelial cells. Immunoprecipitation was carried out using a GFP trap as described in materials and methods. GP129 mutants (NRD13 and GP129UL128) were detected by a myc epitope tag. (i) PC formation assay using gHGFP and other components of the PC and GP129 mutant (NRD13) and GFP trap IP. Lanes 1, 5, 9, 13 and 17 are total cell lysates analyzed by respective antibodies. Lanes 3, 7, 11, 15 and 19 are immunoprecipitation tracks using GFP trap . Lanes 2, 6, 10, 14, 18 are flow through wash tracks prior to immunoprecipitation. Lanes 4, 8, 12, 16 and 20 are mock cell lysate. Samples were assayed for: gH (anti-GFP), lanes 1–4; gL (anti-mCherry), lanes 5–8; GP131 (anti-HA), lanes 9–12; GP133 (anti-FLAG), lanes 13–16; and GP129 mutant NRD13 (anti-myc), lanes 17–20. Secondary antibody was anti-mouse IgG-HRP. (ii) PC formation assay using gHGFP and other components of the PC and GP129 mutant (GP129UL128) and GFP trap IP. Lanes as described for (i) except lanes 17–20 GP129UL128 mutant blot.

Article Snippet: Custom antibodies to GPCMV gH, IE2 and GP131 were generated by Genescript to specific peptide sequences (rabbit polyclonal) or to purified recombinant ( E . coli ) protein (mouse monoclonal).

Techniques: Expressing, Transfection, Immunoprecipitation, Tube Formation Assay, Mutagenesis

Journal: PLoS Pathogens

Article Title: A Homolog Pentameric Complex Dictates Viral Epithelial Tropism, Pathogenicity and Congenital Infection Rate in Guinea Pig Cytomegalovirus

doi: 10.1371/journal.ppat.1005755

Figure Lengend Snippet: GP129, GP131, GP133 and gO were evaluated for an ability to form triplex complexes with gH and gL. Transient expression of epithelial cells with gHGFP, gLmCherry, GP129myc, GP131HA and GP133FLAG was as described in materials and methods. Evaluation of triplex formation was by cellular colocalization or by GFP trap immunoprecipitation assay. A-E. gHGFP/gLmCherry/GP129myc triplex formation. Panels A-D, transient expression of individual proteins in epithelial cells and co-localization shown in merged image (D). Western blot of triplex immunoprecipitation (E). Lanes 1, 4 and 7 (total cell lysate). Lanes 3, 6 and 9 (IP). Lanes 2, 5 and 8 (flow through wash). F-J. gHGFP/gLmCherry/GP131HA triplex formation. Panels F-H, transient expression of individual proteins in epithelial cells and co-localization shown in merged image (I). Western blot of triplex immunoprecipitation (J) as described for E except lanes 7–9 GP131HA western. K-O. gHGFP/gLmCherry/GP133FLAG triplex formation. Panels K-M, transient expression of individual proteins in epithelial cells and co-localization shown in merged image (N). Western blot of triplex immunoprecipitation (O) as described for E except lanes 7–9 GP133FLAG western. P-T. gHGFP/gLmCherry/gOFLAG triplex formation. Panels P-R, transient expression of individual proteins in epithelial cells and co-localization shown in merged image (S). Western blot of triplex immunoprecipitation (T) as described for E except lanes 7–9 gOdelFLAG. Cellular co-localization merged figures (D, K, N and S) include DAPI co-stained cells. GP129myc, GP131HA, GP133FLAG and gOdelFLAG detected by primary anti-epitope antibody and secondary anti-mouseIgG-Cy5 (immunofluorescence) and anti-mouseIgG-HRP (western blot). Both gHGFP and gLmCherry were detected by fluorescence (cell localization) and specific epitope antibody (western). Panels E, J, O and P western blots. Lanes: 1, 4 and 7 total cell lysate; 2, 5 and 8 wash flow through; 3, 6 and 9 immunoprecipitation.

Article Snippet: Custom antibodies to GPCMV gH, IE2 and GP131 were generated by Genescript to specific peptide sequences (rabbit polyclonal) or to purified recombinant ( E . coli ) protein (mouse monoclonal).

Techniques: Expressing, Immunoprecipitation, Western Blot, Staining, Immunofluorescence, Fluorescence

Journal: PLoS Pathogens

Article Title: A Homolog Pentameric Complex Dictates Viral Epithelial Tropism, Pathogenicity and Congenital Infection Rate in Guinea Pig Cytomegalovirus

doi: 10.1371/journal.ppat.1005755

Figure Lengend Snippet: (i) Structure of parental BAC virus (NRD13) and modified mutant GP129FRT GPCMV. Modified mutant encodes an ectopic GP129 (myc tagged) cDNA under SV40 promoter control in the GP25/GP26 intergenic locus as described in materials and methods . Arrows (right) indicate virus tropism to epithelial cells: red, no tropism; green, tropism. (ii) Western blot analysis of sucrose gradient purified GP129FRT. Purified virus was evaluated for the presence of structural proteins present in the viral membrane by western blot analysis: gB (detected by mouse anti-gB), lanes 1–3; gH (rabbit anti-gH), lanes 4–6; GP131 (mouse anti-GP131), lanes 7–9; GP129 (mouse anti-myc), lanes 10–12. Additionally, a control GFP protein (mouse anti-GFP) expressed by the virus was also evaluated (lanes 13–15). Secondary antibodies were either anti-mouse IgG/HRP or anti-rabbit IgG/HRP. Lanes: 1, 4, 7, 10 and 13 total cell lysate (GP129FRT); 2, 5, 8, 11 and 14 total cell lysate (uninfected); 3, 6, 9, 12, and 15 (purified virus particle). Equivalent protein loading was determined by Bradford assay.

Article Snippet: Custom antibodies to GPCMV gH, IE2 and GP131 were generated by Genescript to specific peptide sequences (rabbit polyclonal) or to purified recombinant ( E . coli ) protein (mouse monoclonal).

Techniques: Virus, Modification, Mutagenesis, Control, Western Blot, Purification, Membrane, Bradford Assay