Cobaias como modelo para teste de vacinas inativadas contra o herpesvírus bovino tipo 1 e o vírus da diarréia viral bovina Guinea pigs as a model test of bovine herpesvirus type 1 and bovine viral diarrhea virus inactivated vaccines

O presente trabalho relata a avaliação de cobaias como modelo para testes de imunogenicidade de vacinas inativadas contra o herpesvírus bovino tipo 1 (BoHV-1) e o vírus da diarréia viral bovina (BVDV). Para isso, cobaias (n=60) e bovinos (n=10) foram imunizados duas vezes, com intervalo de 28 dias, com uma vacina experimental contendo antígenos dos dois vírus, e testados para anticorpos neutralizantes 28 dias após a segunda dose. Os bovinos foram vacinados com a dose recomendada para a espécie (5mL); as cobaias foram distribuídas em seis grupos e imunizadas com doses fracionadas (0,005mL a 1,6mL). Os grupos de cobaias imunizadas com doses equivalentes a 1/16 (0,320mL) e 1/8 (0,640mL) da dose bovina desenvolveram títulos médios geométricos (GMTs) de 6,46 e 7,56, respectivamente, estatisticamente semelhantes aos dos bovinos (GMT=8) (P>0,05). Uma alta correlação dose-resposta (R²=0,95) foi observada entre as doses vacinais e os títulos de anticorpos neutralizantes anti-BoHV-1 nos grupos de cobaias. Por outro lado, não foi possível o estabelecimento de uma dose vacinal que induzisse em cobaias uma resposta neutralizante anti-BVDV em níveis semelhantes à induzida em bovinos. Apenas as cobaias imunizadas com as doses maiores (0,640 e 1,6mL) desenvolveram títulos neutralizantes de magnitude moderada (GMTs de 8 e 9, respectivamente), porém estatisticamente inferiores ao GMT dos bovinos (GMT=34,9) (PThe present study reports the use of guinea pigs as a model to study the immunogenicity of bovine herpesvirus type 1 (BoHV-1) and bovine viral diarrhea virus (BVDV) inactivated vaccines. To this purpose, guinea pigs (60) and calves (10) were immunized twice with a 28 day interval with an experimental vaccine containing antigens of both viruses and tested for virus neutralizing (VN) antibodies 28 days after the second dose. Calves were immunized with the recommended dose (5mL), while the guinea pigs were distributed in six groups and immunized with fractionated doses (0.005 to 1.6mL). Guinea pigs immunized with 1/16 (0.320mL) and 1/8 (0.640mL) of the bovine dose developed VN titers (GMTs) of 6.46 and 7.56, respectively, which were equivalent to the titers developed by calves (GMT=8) (P>0.05). A high correlation (R²=0.95) was observed between the antigen dose and the VN titer developed by all guinea pig groups. On the other hand, it was not possible to establish an antigen dose that induces in guinea pigs a serological response to BVDV equivalent to that induced in calves. Only the two groups given the highest antigen doses developed a consistent anti - BVDV neutralizing response, yet with VN titers (GMT= 8 and 9, respectively) significantly lower (P<0.05) than that induced in calves (GMT=34.9). These results demonstrate that guinea pigs may be used as a model to test the immunogenicity of BoHV-1 inactivated vaccines. Volumes between 1/8 and 1/16 of the bovine dose induce in these animals a VN antibody response of equivalent magnitude to that induced in calves

Ablação cirúrgica dos bulbos olfatórios em coelhos: modelo para estudos de patogenia de infecções por vírus neurotrópicos

Coelhos têm sido utilizados como modelo para o estudo da neuropatogenia da infecção pelo herpesvírus bovino tipo 5 (BHV-5), um importante agente de doença neurológica em bovinos. O sistema olfatório tem sido apontado como a principal via de acesso do vírus ao cérebro após replicação na cavidade nasal. Para investigar a importância da via olfatória na patogenia da infecção neurológica pelo BHV-5, foi elaborada e avaliada uma técnica operatória de craniotomia transfrontal para remoção dos bulbos olfatórios (BOs), definindo-se as órbitas como referência anatômica. Foram utilizados 45 coelhos com 30 dias de idade, sendo 23 submetidos à ablação cirúrgica dos BOs e posteriormente inoculados pela via intranasal (IN) ou no saco conjuntival (IC) com o BHV-5. Após incisões de pele, tecido subcutâneo e periósteo, a craniotomia foi realizada em um ponto eqüidistante entre os cantos mediais dos olhos, com uma broca sulcada de 3mm acoplada a uma perfuratriz elétrica de baixa rotação. A remoção dos BOs foi realizada com uma sonda uretral ndegrees6 acoplada a um aspirador. O estudo macroscópico de três animais após a cirurgia comprovou que o procedimento foi eficiente na remoção total dos BOs. Isso também foi comprovado pela interrupção do acesso do vírus ao córtex cerebral: apenas um animal (1/11 ou 9,1%) no grupo submetido à ablação dos BOs com inoculação IN desenvolveu enfermidade neurológica, contra 100% (10/10) dos coelhos controle. Conclui-se que a técnica de craniotomia transfrontal utilizando a órbita como referência anatômica permite o acesso adequado para localização e remoção dos BOs e pode ser utilizada em estudos de patogenia de infecções por vírus neurotrópicos que exijam a interrupção completa da via olfatória

A parapoxviral virion protein inhibits NF-κB signaling early in infection - Fig 7

<p>(A) Detection of ORFV073 in virions. OFTu cells were infected with OV-IA82Δ073 or OV-IA82RV073^Flag and harvested at 90–95% cytopathic effect. Supernatants and pellets from infected cultures were purified for extracellular enveloped virus (EEV) and intracellular mature virus (IMV) as described in Material and Methods. Whole cell lysates (10 μg) from mock and OV-IA82RV073^Flag infected cells (MOI = 10) (24 h p.i) and purified OV-IA82Δ073 and OV-IA82RV073^Flag virion proteins (10 μg) were resolved by SDS-PAGE, blotted to the nitrocellulose membrane and probed with antibodies against flag or structural protein ORFV086. # and ## denote 30 kDa and a doublet 40 kDa ORFV073 bands in EEV fraction, respectively. (B) Effect of translation inhibition on ORFV073-mediated block of NF-κB-p65 nuclear translocation. OFTu cells were pre-treated with the protein synthesis inhibitor cycloheximide (CHX) (50 μg/ml) for 30 min and then mock infected or infected with OV-IA82, OV-IA82Δ073 or OV-IA82RV073^Flag (MOI = 10) in presence of CHX (50 μg/ml). Cells were fixed at 1 h p.i., incubated with antibody against NF-κB-p65, stained with Alexa Fluor 488-labeled antibody and DAPI, and examined by confocal microscopy. Cells (n = approximately 300) expressing nuclear NF-κB-p65 in each group were counted and results depicted as mean percentage of cells containing nuclear NF-κB-p65 over two independent experiments (*, <i>P<0</i>.<i>05</i>). Result shown in (A) are representative of six independent experiments.</p

Replication characteristics of ORFV073-gene deletion virus.

<p>Primary OFTu cells were infected with wild-type OV-IA82 or deletion mutant OV-IA82Δ073 viruses and virus titers determined at various times p.i. as described in Material and Methods. (A) Multi-step growth curve, MOI 0.1; (B) single-step growth curve, MOI 10. (C) CPE of wild-type OV-IA82 or deletion mutant OV-IA82Δ073 virus infected OFTu cells at 48 h p.i (MOI = 10) (magnification, X20).</p

Effect of ORFV073 expression on TNFα-induced phosphorylation of IKKα/β, IκBα and NF-κB-p65.

<p>(A) HeLa cells stably expressing GFP (GFP/HeLa) or ORFV073-GFP (073GFP/HeLa) were treated with TNFα (20 ng/ml) and harvested at 5, 10 and 15 min post treatment. Whole cell protein extracts (50 μg) were resolved by SDS-PAGE, blotted to nitrocellulose membranes and probed with antibodies against phospho or total IKKα/β, IκBα and NF-κB-p65, and GAPDH and GFP. (B-D) Densitometry of p-IKKα/β, p- IκBα and p-NF-κB-p65 bands, respectively are normalized to the loading control GAPDH. Relative fold inductions across treatments in B-D are shown relative to GFP/HELA (*, <i>P<0</i>.<i>05</i> and **, <i>P<0</i>.<i>01</i>). Results are mean values of three independent experiments.</p

Clustal W alignment of amino acid sequences corresponding to parapoxvirus ORFV073 homologs, squirrellpox virus (SQPV)-084 protein, and murid betaherpesvirus m170 protein.

<p>The ORFV strain IA82 sequence is at the top of the alignment, with amino acid positions indicated by numbers on the right. The predicted nuclear localization signal is shown underlined. Sequences listed below strain IA82 are arranged in order of decreasing % amino acid identity relative to strain IA82. For clarity, only the murid betaherpesvirus m170 domain with homology to parapoxvirus ORFV073 proteins is shown (m170 positions 86–147). Asterisks [*], colons [:], and periods [.] below the alignment indicate fully, strongly, and weakly conserved, residues, respectively. Viruses, strains, and GenBank accession numbers (in parentheses) used for the alignment are as follows: ORFV strains OV-IA82 (AY386263.1), NZ2 (DQ184476.1), B029 (KF837136.1), NA1/11 (KF234407.1), D1701 (HM133903.1), NP (KP010355.1), SJ1 (KP010356.1), OV-SA00 (NC 005336.1), GO (KP010354.1), and YX (KP010353.1); Pseudocowpox virus strain F00.120R [PCPV-F00] (GQ329669.1); Bovine papular stomatitis virus strain BV-AR02 [BPSVar02] (NC 005337.1); Parapoxvirus of the red deer in New Zealand strain HL953 [PPV-RD] (NC 025963.1); Squirrellpox virus strain Red squirrel UK [SQPV-084] (YP_008658509); Murid betaherpesvirus 1 strain N1 [MHV1m170] (CCE57166).</p

Effect of ORFV073 expression on TNFα-induced NF-κB-p65 nuclear translocation.

<p>(A) HeLa cells stably expressing GFP (GFP/HeLa) or ORFV073-GFP fusion protein (073GFP/HeLa) were treated with TNFα (20 ng/ml) and fixed 30 min or 1 h after treatment. Cells were sequentially incubated with anti-NF-κB-p65 and Alexa Fluor 594-labeled antibodies, and stained with DAPI. Shown are representative results of two independent experiments after 30 min TNFα treatment. GFP and 073GFP, Green; NF-κB-p65, red; DAPI, blue. Arrows indicate ORFV073-expressing cells with absence or reduced nuclear NF-κB-p65. (B) Cells (n = approximately 200) from randomly selected fields were scored for nuclear NF-κB-p65 and results are depicted as mean percentage of GFP/073GFP expressing cells containing nuclear NF-κB-p65 at 30 min and 1 h post TNFα induction over two independent experiments (*, P<0.05).</p

Co-immunoprecipitation of ORFV073 with NEMO.

<p>OFTu cells co-transfected with plasmids pcDNA3.1-NEMO, pcDNA/V5-His (control), or pcDNA3.1-NEMO and pcDNA/V5-073His (ORFV073-His) were harvested at 24 h post transfection and nuclear extracts prepared as described in Material and Methods. Nuclear lysates (left) and extracts immunoprecipitated with anti-His (upper right) or anti-NEMO (lower right) antibodies were examined by SDS-PAGE-Western blotting with antibodies directed against proteins indicated on the right. # denote light chain of the IgG antibody. Results are representative of three independent experiments.</p