Congenital lobar emphysema in a kitten.

A five-month-old ragdoll cat presented with severe respiratory signs, unresponsive to medical therapy. Hyperinflation of the right middle lung lobe was diagnosed with radiography and computed tomography. Lung lobectomy following a median sternotomy led to full recovery. Histopathological analysis revealed lobar emphysema and, based on the animal's age, congenital lobar emphysema was considered the most likely diagnosis.

Sensitivity of porcine epidemic diarrhea virus (PEDV) to pH and heat treatment in the presence or absence of porcine plasma.

Emergence of porcine epidemic diarrhea virus (PEDV) resulted in massive neonatal mortality in the North-American and Asian pork industry. Measures to prevent its geographical spread are of utmost importance to safeguard susceptible porcine populations. The major infection route is direct or indirect faecal-oral contact. Adequate biosafety measures should be in place at all levels of the swine production chain, including feed and feed ingredients. Present study aimed to investigate the sensitivity of PEDV to thermal inactivation at neutral and alkaline pH in presence or absence of porcine plasma. Cell culture medium and porcine plasma at different pH (7.2, 9.2, 10.2) and temperature conditions (4 °C, 40 °C, 44 °C, 48 °C) were inoculated to a final titer of 5.5 log10 TCID50 PEDV/ml, incubated for up to 120 min and the residual infectivity was determined by endpoint dilution assay. Irrespective of presence of plasma, PEDV was not sensitive to pH 7.2-10.2 at 4 °C. At moderate temperatures (≥40 °C), both alkaline pH and presence of plasma potentiated thermal inactivation. Inactivation of 8 log10 TCID50/ml plasma within 30 min (8D value<30 min) by moderate pH and temperature would denote potential industrial processing conditions that ensure safety towards PEDV while limiting denaturation of bioactive components. Virus-spiked plasma required heat treatment of 40 °C and alkalinization to pH 9.2 to achieve 8 log10 reduction within such time. At pH 10.2 and 48 °C, the 8D value was 4.6 min in plasma and 15.2 min in MEM. Here we propose heat-alkalinity-time (HAT) pasteurization as a highly efficient method to inactivate PEDV during industrial processing of porcine plasma.

Glycoprotein K specified by herpes simplex virus type 1 is expressed on virions as a Golgi complex-dependent glycosylated species and functions in virion entry.

To facilitate detection of glycoprotein K (gK) specified by herpes simplex virus, a 12-amino-acid epitope tag was inserted within gK domain III. Recombinant virus gKprotC-DIII, expressing the tagged gK, was isolated. This virus formed wild-type plaques and replicated as efficiently as the wild-type KOS virus in Vero cells. Anti-protein C MAb detected high-mannose and Golgi complex-dependent glycosylated gK within cells as well as on purified virions. The gK-null virus DeltagK (gK(-/-)) entered Vero cells substantially more slowly than the wild-type KOS (gK(+/+)), while DeltagK virus grown in complementing VK302 cells (gK(-/+)) entered with entry kinetics similar to those of the KOS virus.

Expression of the enhanced green fluorescent protein by herpes simplex virus type 1 (HSV-1) as an in vitro or in vivo marker for virus entry and replication.

Glycoprotein K (gK) is involved in membrane fusion phenomena during infectious virus production and egress and is an important determinant for neurovirulence. To assess better the in vitro and in vivo roles of gK in virus replication, a recombinant virus was constructed expressing an engineered enhanced green fluorescent protein (EGFP) under the control of the human cytomegalovirus immediate early gene promoter (HCMV-IEP) inserted in place of the gK gene. The EGFP gene insertion was confirmed by diagnostic polymerase chain reaction (PCR), and the presence of the EGFP protein was detected by western immunoblot analysis using anti-GFP monoclonal antibody. Fluorescence microscopy revealed that virus infected cells emitted bright fluorescence when examined using filters for fluorescein. Fluorescence emission was detected as early as 4 h post-infection. Fluorescence intensity increased over time and was stable at late times after infection at which point viral plaques continued to emit bright green fluorescence. The amount of fluorescence emitted by virus infected Vero cells was monitored by fluorescence cytometry using a FACS cytometer. At an MOI of 3, all infected cells emitted strong green fluorescence as quantified by cytometry at 48 h post-infection. The deltagK-EGFP expressing recombinant virus will enable the determination of the role of gK in virus entry and egress as well as the role of gK in the molecular pathogenesis of herpes simplex virus type 1 (HSV-1).

Efficient long-PCR site-specific mutagenesis of a high GC template.

A long PCR method was developed for the efficient site-specific mutagenesis of herpes simplex virus (HSV-1) DNA fragments with high GC content. In this protocol, a PCR product was partially extended first using a cloned DNA fragment. The final mutagenized fragment was produced after a second extension using another PCR product and final amplification using external primers. The sequential use of two extension reactions increased the predicted frequency of the engineered mutation in the final product to 100%. This method was used to generate a mutated glycoprotein K (gK) gene specified by HSV-1. A recombinant virus that carried the mutated gK gene caused extensive cell fusion of infected cells.