Journal: Journal of Virology

Article Title: Temperature-Sensitive Live-Attenuated Canine Influenza Virus H3N8 Vaccine

doi: 10.1128/JVI.02211-16

Figure Lengend Snippet: Effect of temperature on the polymerase activity of H3N8 LACIV. (A) Schematic representation of segments 1 (PB2) and 2 (PB1) of WT (black) and LACIV (gray) H3N8 CIV. Amino acid substitutions N265S (PB2) and K391E, E581G, and A661T (PB1) to generate the H3N8 LACIV are indicated. (B) Minigenome activity. MDCK cells (12-well plate format, 3 × 105 cells/well, triplicates) were transiently cotransfected with 250 ng of ambisense pDZ expression plasmids encoding the minimal requirements for viral genome replication and gene transcription (PB2, PB1, PA, and NP), together with 500 ng of a vRNA-like expression plasmid encoding Gaussia luciferase (Gluc) under the control of the canine polymerase I promoter (cpPol-I Gluc), and 100 ng of a pCAGGS Cypridina luciferase (Cluc) plasmid to normalize transfection efficiencies. After transfection, cells were placed at 33, 37, or 39°C, and viral replication and transcription was evaluated 24 h later by luminescence (Gluc). Gluc activity was normalized to that of Cluc. The data represent means and SD. Normalized reporter expression is relative to that in the absence of pDZ NP plasmid. Data were represented as relative activity considering WT H3N8 polymerase activity at each temperature as 100%. *, P < 0.05 (Student t test).

Article Snippet: Alternatively, 100 μl of a commercially available inactivated H3N8 CIV vaccine (Nobivac; Merck Animal Health) or inactivated H3N2 CIV vaccine (Zoetis) were inoculated i.m.

Techniques: Activity Assay, Expressing, Plasmid Preparation, Luciferase, Transfection

Journal: Journal of Virology

Article Title: Temperature-Sensitive Live-Attenuated Canine Influenza Virus H3N8 Vaccine

doi: 10.1128/JVI.02211-16

Figure Lengend Snippet: Characterization of H3N8 LACIV in vitro: MDCK cells (12-well plate format, 3 × 105 cells/well, triplicates) were infected (MOI of 0.001) with WT (black diamonds) and LACIV (gray squares) H3N8 CIVs and incubated at 33°C (A), 37°C (B), and 39°C (C). TCS were collected at 12, 24, 48, 72, and 96 h p.i., and the viral titers were determined by immunofocus assay (FFU/ml). Data represent the means and SD of the results determined in triplicate. Dotted lines indicate the limit of detection (200 FFU/ml). *, P < 0.05 (Student t test).

Article Snippet: Alternatively, 100 μl of a commercially available inactivated H3N8 CIV vaccine (Nobivac; Merck Animal Health) or inactivated H3N2 CIV vaccine (Zoetis) were inoculated i.m.

Techniques: In Vitro, Infection, Incubation

Journal: Journal of Virology

Article Title: Temperature-Sensitive Live-Attenuated Canine Influenza Virus H3N8 Vaccine

doi: 10.1128/JVI.02211-16

Figure Lengend Snippet: Attenuation of H3N8 LACIV in vivo. Female 5- to-7-week-old C57BL/6 WT mice (n = 6) were infected i.n. with 105 PFU of WT or LACIV H3N8 CIVs. Three mice were sacrificed at days 2 (black) and 4 (gray) p.i., and the lungs were harvested for virus titrations using an immunofocus assay (FFU/ml). Data represent the means and SD. Dotted line indicate limit of detection (200 FFU/ml). ND, virus not detected.

Article Snippet: Alternatively, 100 μl of a commercially available inactivated H3N8 CIV vaccine (Nobivac; Merck Animal Health) or inactivated H3N2 CIV vaccine (Zoetis) were inoculated i.m.

Techniques: In Vivo, Infection

Journal: Journal of Virology

Article Title: Temperature-Sensitive Live-Attenuated Canine Influenza Virus H3N8 Vaccine

doi: 10.1128/JVI.02211-16

Figure Lengend Snippet: Immunogenicity of H3N8 LACIV: Female 5- to-7-week-old C57BL/6 WT mice (n = 6) were vaccinated i.n. with 103 PFU of WT or LACIV H3N8 CIVs. Mice mock (PBS) vaccinated or vaccinated i.m. with 100 μl of an H3N8 CIV IIV (Nobivac) were used as internal controls. (A) Induction of humoral responses. At 14 days postvaccination, mice were bled, and sera were evaluated for the presence of total IgG antibodies against H3N8 CIV proteins using cell extracts of MDCK-infected cells by ELISA. MDCK mock-infected cell extracts were used to evaluate the specificity of the antibody response. OD, optical density. Data represent the means ± the SD of the results for six individual mice. *, Nobivac versus LACIV; **, WT versus LACIV; ***, WT versus Nobivac (P < 0.05 using the Student t test). (B) HAI titers. Sera from immunized mice were evaluated by HAI using four HAU of WT H3N8 CIV and 2-fold serial dilutions of the indicated sera. ND, not detected. *, WT versus LACIV or Nobivac; or *, LACIV versus Nobivac (P < 0.05 using the Student t test).

Article Snippet: Alternatively, 100 μl of a commercially available inactivated H3N8 CIV vaccine (Nobivac; Merck Animal Health) or inactivated H3N2 CIV vaccine (Zoetis) were inoculated i.m.

Techniques: Infection, Enzyme-linked Immunosorbent Assay

Journal: Journal of Virology

Article Title: Temperature-Sensitive Live-Attenuated Canine Influenza Virus H3N8 Vaccine

doi: 10.1128/JVI.02211-16

Figure Lengend Snippet: CD8 T cell response induced by H3N8 LACIV. Female 5- to-7-week-old C57BL/6 WT mice (n = 4) were vaccinated i.n. with 103 PFU of WT or LACIV H3N8 CIVs. Mice mock (PBS) vaccinated or vaccinated i.m. with 100 μl of an H3N8 CIV IIV (Nobivac) were used as internal controls. At 10 days p.i., the lungs (A) and spleen (B) were extracted, and the cells were prepared for flow cytometry. Live CD8 T cells specific for NP or PA tetramers were counted. The data represent the means ± the SD of the results for four individual mice. *, WT versus LACIV, Nobivac, or PBS; or *, LACIV versus Nobivac or PBS (P < 0.05 using the Student t test).

Article Snippet: Alternatively, 100 μl of a commercially available inactivated H3N8 CIV vaccine (Nobivac; Merck Animal Health) or inactivated H3N2 CIV vaccine (Zoetis) were inoculated i.m.

Techniques: Flow Cytometry

Journal: Journal of Virology

Article Title: Temperature-Sensitive Live-Attenuated Canine Influenza Virus H3N8 Vaccine

doi: 10.1128/JVI.02211-16

Figure Lengend Snippet: Protection efficacy of H3N8 LACIV against homologous viral challenge. Female 5- to-7-week-old C57BL/6 WT mice (n = 6) were vaccinated i.n. with 103 PFU of WT or LACIV H3N8 CIVs. Mice mock (PBS) vaccinated or vaccinated i.m. with 100 μl of an H3N8 CIV IIV (Nobivac) were used as internal controls. At 15 days postvaccination, mice were challenged i.n. with 105 PFU of WT H3N8 CIV. To evaluate viral replication, mice were euthanized at days 2 (n = 3, black) and 4 (n = 3, gray) postchallenge, and the lungs were harvested, homogenized, and used to quantify viral titers by immunofocus assay (FFU/ml). The dotted line indicates the limit of detection (200 FFU/ml). ND, virus not detected. Data represent the mean ± the SD. *, P < 0.05 using the Student t test.

Article Snippet: Alternatively, 100 μl of a commercially available inactivated H3N8 CIV vaccine (Nobivac; Merck Animal Health) or inactivated H3N2 CIV vaccine (Zoetis) were inoculated i.m.

Techniques:

Journal: Journal of Virology

Article Title: Temperature-Sensitive Live-Attenuated Canine Influenza Virus H3N8 Vaccine

doi: 10.1128/JVI.02211-16

Figure Lengend Snippet: Comparison of WT and LACIV H3N8 CIV infection phenotypes in canine tracheal explants. (A) Histological features of dog tracheal explants infected with 200 PFU of H3N8 WT CIV or H3N8 LACIV or mock-infected with infection media. Lesions are shown in sections stained with H&E. (B) CIV H3N8-infected cells were detected by immunostaining for the viral NP, and positive cells are stained in brown. For both panels A and B, pictures are representatives of three independent experiments, and the black horizontal bars represent 20 μm. (C) Graphical representation of the bead clearance average time of CIV- or mock-infected dog tracheal explants for three independent experiments. Data represent the means ± the SD. ns, P > 0.05 (D1, LACIV versus Mock); *, P < 0.05 (D1, WT versus Mock); **, P < 0.01 (D3, LACIV versus Mock); ***, P < 0.001 (D3, WT versus Mock); ****, P < 0.0001 (D5, LACIV and WT versus Mock). (D) Average viral replication of H3N8 LACIV and H3N8 WT CIV in canine tracheal explants from three independent experiments. Data represent the means ± the SD. The dotted line indicates the limit of detection (20 FFU/ml).

Article Snippet: Alternatively, 100 μl of a commercially available inactivated H3N8 CIV vaccine (Nobivac; Merck Animal Health) or inactivated H3N2 CIV vaccine (Zoetis) were inoculated i.m.

Techniques: Infection, Staining, Immunostaining

Journal: Journal of Virology

Article Title: Temperature-Sensitive Live-Attenuated Canine Influenza Virus H3N8 Vaccine

doi: 10.1128/JVI.02211-16

Figure Lengend Snippet: Immunogenicity and protection efficacy of H3N8 LACIV against heterologous H3N2 CIV challenge. Female 5- to-7-week-old C57BL/6 WT mice were vaccinated i.n. with 103 PFU of WT and LACIV H3N8 CIVs. Mice mock (PBS) vaccinated or vaccinated i.m. with 100 μl of the H3N8 (Nobivac) and an H3N2 CIV IIV (Zoetis) were used as internal controls. (A to C) Antibody cross-reactivity against the heterologous CIV H3N2. At 14 days postvaccination, mice were bled, and sera were evaluated by ELISA for total IgG antibodies against H3N2 CIV proteins using cell extracts of MDCK-infected cells (A). Mock-infected MDCK cell extracts were used to evaluate the specificity of the antibody response. OD, optical density. Data represent the means ± the SD of the results for six individual mice. *, Nobivac versus LACIV; **, WT versus LACIV; or ***, WT versus Nobivac (P < 0.05 using the Student t test). Specific antibody response against recombinant HA (B) and NA (C) proteins from H3N2 CIV were evaluated by ELISA. Data represent the means ± the SD of the results for pooled serum samples. *, WT versus LACIV (P < 0.05 using the Student t test). (D) Protection efficacy of H3N8 LACIV against heterologous H3N2 CIV challenge. At 15 days postvaccination, mice were challenged i.n. with 105 PFU of WT H3N2 CIV. To evaluate WT H3N2 CIV replication, mice were sacrificed at days 2 (n = 3, black) and 4 (n = 3, gray) postchallenge, and the lungs were harvested, homogenized, and used to evaluate the presence of virus by immunofocus assay (FFU/ml). The dotted line indicates the limit of detection (200 FFU/ml). ND, virus not detected. Data represent means ± the SD. *, P < 0.05 using Student t test.

Article Snippet: Alternatively, 100 μl of a commercially available inactivated H3N8 CIV vaccine (Nobivac; Merck Animal Health) or inactivated H3N2 CIV vaccine (Zoetis) were inoculated i.m.

Techniques: Enzyme-linked Immunosorbent Assay, Infection, Recombinant