Binding and entry characteristics of porcine circovirus 2 in cells of the porcine monocytic line 3D4/31.

&lt;p&gt;Porcine circovirus 2 (PCV2) is associated with post-weaning multisystemic wasting syndrome and reproductive problems in pigs. Cells of the monocyte/macrophage lineage are important target cells in PCV2-infected pigs, but the method of binding and entry of PCV2 into these cells is unknown. Therefore, binding and entry of PCV2 to the porcine monocytic cell line 3D4/31 were studied by visualization of binding and internalization of PCV2 virus-like particles (VLPs) by confocal microscopy and chemical inhibition of endocytic pathways (clathrin- and caveolae-mediated endocytosis and macropinocytosis), followed by evaluation of the level of PCV2 infection. It was shown that PCV2 VLPs bound to all cells, with maximal binding starting from 30 min post-incubation. Bound PCV2 VLPs were internalized in 47+/-5.0 % of cells. Internalization was continuous, with 70.5+/-9.7 % of bound PCV2 VLPs internalized at 360 min post-incubation. Internalizing PCV2 VLPs co-localized with clathrin. PCV2 infection was decreased significantly by chemical inhibitors that specifically blocked (i) actin-dependent processes, including cytochalasin D (75.5+/-7.0 % reduction) and latrunculin B (71.0+/-3.0 % reduction), and (ii) clathrin-mediated endocytosis, including potassium depletion combined with hypotonic shock (50.2+/-6.3 % reduction), hypertonic medium (56.4+/-5.7 % reduction), cytosol acidification (59.1+/-7.1 % reduction) and amantadine (52.6+/-6.7 % reduction). Inhibiting macropinocytosis with amiloride and caveolae-dependent endocytosis with nystatin did not decrease PCV2 infection significantly. PCV2 infection was reduced by the lysosomotropic weak bases ammonium chloride (47.0+/-7.9 % reduction) and chloroquine diphosphate (49.0+/-5.6 % reduction). Together, these data demonstrate that PCV2 enters 3D4/31 cells predominantly via clathrin-mediated endocytosis and requires an acidic environment for infection.&lt;/p&gt;</p

Binding and entry characteristics of porcine circovirus 2 in cells of the porcine monocytic line 3D4/31.

&lt;p&gt;Porcine circovirus 2 (PCV2) is associated with post-weaning multisystemic wasting syndrome and reproductive problems in pigs. Cells of the monocyte/macrophage lineage are important target cells in PCV2-infected pigs, but the method of binding and entry of PCV2 into these cells is unknown. Therefore, binding and entry of PCV2 to the porcine monocytic cell line 3D4/31 were studied by visualization of binding and internalization of PCV2 virus-like particles (VLPs) by confocal microscopy and chemical inhibition of endocytic pathways (clathrin- and caveolae-mediated endocytosis and macropinocytosis), followed by evaluation of the level of PCV2 infection. It was shown that PCV2 VLPs bound to all cells, with maximal binding starting from 30 min post-incubation. Bound PCV2 VLPs were internalized in 47+/-5.0 % of cells. Internalization was continuous, with 70.5+/-9.7 % of bound PCV2 VLPs internalized at 360 min post-incubation. Internalizing PCV2 VLPs co-localized with clathrin. PCV2 infection was decreased significantly by chemical inhibitors that specifically blocked (i) actin-dependent processes, including cytochalasin D (75.5+/-7.0 % reduction) and latrunculin B (71.0+/-3.0 % reduction), and (ii) clathrin-mediated endocytosis, including potassium depletion combined with hypotonic shock (50.2+/-6.3 % reduction), hypertonic medium (56.4+/-5.7 % reduction), cytosol acidification (59.1+/-7.1 % reduction) and amantadine (52.6+/-6.7 % reduction). Inhibiting macropinocytosis with amiloride and caveolae-dependent endocytosis with nystatin did not decrease PCV2 infection significantly. PCV2 infection was reduced by the lysosomotropic weak bases ammonium chloride (47.0+/-7.9 % reduction) and chloroquine diphosphate (49.0+/-5.6 % reduction). Together, these data demonstrate that PCV2 enters 3D4/31 cells predominantly via clathrin-mediated endocytosis and requires an acidic environment for infection.&lt;/p&gt;</p

Isolamento e caracterização do vírus da influenza pandêmico H1N1 em suínos no Brasil

A infecção causada pelo vírus Influenza A (IAV) é endêmica em suínos no mundo inteiro. O surgimento da pandemia de influenza humana pelo vírus A/H1N1 (pH1N1) em 2009 levantou dúvidas sobre a ocorrência deste vírus em suínos no Brasil. Durante o desenvolvimento de um projeto de pesquisa do vírus de influenza suína em 2009-2010, na Embrapa Suínos e Aves (CNPSA), foi detectado em um rebanho de suínos em Santa Catarina, Brasil, um surto de influenza altamente transmissível causado pelo subtipo viral H1N1. Este vírus causou uma doença leve em suínos em crescimento e em fêmeas adultas, sem mortalidade. Tres leitões clinicamente afetados foram eutanasiados. As lesões macroscópicas incluiam consolidação leve a moderada das áreas cranioventrais do pulmão. Microscopicamente, as lesões foram caracterizadas por bronquiolite necrosante obliterativa e pneumonia broncointersticial. A imunohistoquímica, utilizando um anticorpo monoclonal contra a nucleoproteína do vírus influenza A, revelou marcação positiva no núcleo das células epiteliais bronquiolares. O tecido pulmonar de três leitões e os suabes nasais de cinco fêmeas e quatro leitões foram positivos para influenza A pela RT-PCR. O vírus influenza foi isolado de um pulmão, mais tarde sendo confirmado pelo teste de hemaglutinação (título HA 1:128) e por RT-PCR. A análise das seqüências de nucleotídeos dos genes da hemaglutinina (HA) e proteína da matriz (M) revelou que o vírus isolado foi consistente com o vírus pandêmico A/H1N1/2009 que circulou em humanos no mesmo período. Este é o primeiro relato de um surto de influenza causado pelo vírus pandêmico A/H1N1 em suínos no Brasil