Porcine circovirus type 2 morphogenesis in a clone derived from the l35 lymphoblastoid cell line.

Porcine circovirus type 2 (PCV2) is the essential infectious agent of post-weaning multisystemic wasting syndrome (PMWS), one of the most important diseases of swine. Although several studies have described different biological properties of the virus, some aspects of its replication cycle, including ultrastructural alterations, remain unknown. The aim of the present study was to describe for the first time a complete morphogenesis study of PCV2 in a clone of the lymphoblastoid L35 cell line at the ultrastructural level using electron microscopy techniques. Cells were infected with PCV2 at a multiplicity of infection of 10 and examined at 0, 6, 12, 24, 48, 60 and 72h post-infection. PCV2 was internalized by endocytosis, after which the virus aggregated in intracytoplasmic inclusion bodies (ICIs). Subsequently, PCV2 was closely associated with mitochondria, completing a first cytoplasmic phase. The virus entered the nucleus for replication and virus assembly and encapsidation occurred with the participation of the nuclear membrane. Immature virions left the nucleus and formed ICIs in a second cytoplasmic phase. The results suggest that at the end of the replication cycle (between 24 and 48h), PCV2 was released either by budding of mature virion clusters or by lysis of apoptotic or dead cells. In conclusion, the L35-derived clone represents a suitable in-vitro model for PCV2 morphogenesis studies and characterization of the PCV2 replication cycle.

Subcellular immunolocalization of porcine circovirus type 2 (PCV2) in lymph nodes from pigs with post-weaning multisystemic wasting syndrome (PMWS).

Post-weaning multisystemic wasting syndrome (PMWS) is one of the most significant porcine diseases worldwide. The causative agent is porcine circovirus type 2 (PCV2), the smallest virus known to infect animals. Data related to the structural and ultrastructural aspects of this infectious disease are sparse and there is little knowledge of the subcellular localization of PCV2 and its replication in the tissues of pigs naturally affected by PMWS. The present study describes the cellular localization of PCV2 in the lymph nodes of pigs affected by PMWS by application of immunolabelling techniques for light and transmission electron microscopy (TEM). PCV2 particles were exclusively detected in histiocytes. Ultrastructural alterations including marked dilatation of rough endoplasmic reticulum and swelling of mitochondria were associated with PCV2-labelled intracytoplasmic inclusions (ICIs) with recognizable virions. Within the ICIs icosahedral virus-like particles were specifically labelled with a PCV2 capsid antibody, whereas particles with a granular appearance were not labelled. Colocalization studies with confocal microscopy and double immunolabelling with TEM indicated a close relationship between virus and the mitochondria, suggesting that these organelles may play an important role in the replication of PCV2. The present findings further support the hypothesis that virus replicates within the histiocytes of lymph nodes.

Expression of KIT receptor in feline cutaneous mast cell tumors.

Twenty-seven feline cutaneous mast cell tumors (MCTs) were selected for this retrospective study. Samples were routinely processed and stained with hematoxylin and eosin (HE) and toluidine blue, and tumors were classified as well-differentiated (19/27), atypical or poorly granulate (7/27), and pleomorphic (1/27). Immunohistochemistry to detect KIT protein was performed on all samples. The immunoreactivity was recorded by distribution within the tumor, cellular location, and intensity. Well-differentiated MCTs were predominantly characterized by diffuse cytoplasmic (8/19) and membranous stain (7/19); a diffuse distribution of KIT positive cells was displayed in most of these tumors as well (15/19). Atypical MCTs showed diffuse distribution of labeled cells (4/7), and diffuse cytoplasm immunostaining was seen most (5/7). The pleomorphic MCT showed diffuse cytoplasmic KIT stain, with moderate labeling intensity, typically displaying focal distribution in deeper areas of the neoplasm. According to the results, there was no correlation between the type of MCTs and KIT expression, although the use of feline KIT immunohistochemistry could be useful to assess the mast cell origin.

Ultrastructural findings in lymph nodes from pigs suffering from naturally occurring postweaning multisystemic wasting syndrome.

The aims of this study were to evaluate ultrastructural lesions in lymph nodes from postweaning multisystemic wasting syndrome (PMWS)-affected pigs and to correlate these alterations with detection of viral-like particles (VLPs). Samples of lymph nodes were taken from 4 PMWS-affected pigs and 2 healthy animals and processed by transmission electron microscopy. Significant ultrastructural alterations were only noted in PMWS-affected pigs, mainly in histiocytes and rarely in other cell types. Histiocytes showed severe swelling and proliferation of mitochondria, and proliferation and dilation of rough endoplasmic reticulum and Golgi complex. Infected histiocytes contained large numbers of intracytoplasmic inclusion (ICI) bodies with VLPs; some histiocytes also had intranuclear inclusions (INIs). Small inclusions were surrounded by double membrane, with a granular appearance or containing paracrystalline arrays; icosahedral VLPs were 8-17 nm in diameter. Large ICIs were double-membrane bounded or not and contained VLPs usually forming paracrystalline arrays. ICIs were often found next to mitochondria with severe swelling, and also inside them. INIs were not surrounded by membranes and contained virions of 10-13 nm diameter. Lymphocyte depletion was a striking finding of lymph nodes from PMWS-affected pigs. The inclusion bodies containing VLPs referred to in the present study should be classified as viral factories, suggesting that viral replication is probably a frequent event in macrophages, in which mitochondria might play a role.