Characterisation of the differences between hepatitis C virus genotype 3 and 1 glycoproteins

Sequence variation in the envelope E1 and E2 glycoproteins of hepatitis C virus (HCV) could account for differences in disease pathogenesis in patients infected with different genotypes. A cDNA encoding the structural region of the hepatitis C polyprotein was constructed to match the majority sequence of viral RNA extracted from a patient infected with genotype 3a (designated strain HCV3a-Gla). The principal differences predicted between E2 of HCV3a-Gla and the corresponding H77c genotype 1a protein were that the former contained six more amino acids (361 vs. 355), but it had one fewer glycosylation site. Expression studies showed that, in common with the H77c glycoproteins, E1 and E2 from HCV3a-Gla localised to the endoplasmic reticulum (ER) membrane in both Huh-7 and BHK tissue culture cells and interacted to form native complexes. Analysis of the cross-reactivity of antibodies raised against glycoproteins of genotype 1a strains showed that three of five monoclonal antibodies that recognise linear epitopes were able to detect E2 from strain HCV3a-Gla. However, neither conformational E2 antibodies nor antibodies raised against E1 were able to detect the HCV3a-Gla glycoproteins. In receptor binding assays, E2 of HCV3a-Gla consistently failed to bind CD81, a putative cell receptor for HCV. Absence of binding to CD81 and lack of recognition by most antibodies raised to genotype 1a glycoproteins indicate important differences between these glycoproteins representative of genotypes 3a and 1a. These may be pertinent to the differences in response to interferon therapy and the prevalence of steatosis reported in patients infected with these genotypes.     

A new isolate of beak and feather disease virus from endemic wild red-fronted parakeets (Cyanoramphus novaezelandiae) in New Zealand

Psittacine beak and feather disease (PBFD) is a viral disease distributed worldwide with a potentially critical impact on many rare parrots. While efforts have been made to determine its prevalence in wild and captive psittacines, only limited work has been done to document complete genomes of its causative agent, beak and feather disease virus (BFDV). Here, we describe five full genomes of BFDV isolated from wild specimens of an endemic New Zealand parrot, the red-fronted parakeet (Cyanoramphus novaezelandiae). The isolates share >99% nucleotide similarity amongst themselves and ~91–92% similarity to BFDV isolates from southern Africa, Europe and Australia. A maximum-likelihood (ML) phylogenetic tree including 42 other full-genome sequences indicated that the five isolates from red-fronted parakeets represent an undescribed genotype of BFDV. These isolates are evolutionarily most closely related to the Cacatuini isolates from Thailand and the Lorinae isolates from Australia in the rep gene ML tree; however, in the cp ML tree, the evolutionary relationship is closer to viruses found in the Psittacini.     

In vitro hemocompatibility testing of UV-modified hyaluronan hydrogels

Hydrogels (hylans) based on cross-linked hyaluronan (HA) are potentially good biomaterials for vascular tissue engineering applications because they are highly non-antigenic and -immunogenic. To facilitate surface endothelialization, vital to vascular deployment, we irradiated the gel surface with low wavelength UV light. This process micro-textures the smooth gel surface to provide sites for cell anchorage and causes limited scission of native long-chain HA yielding smaller fragments that elicit an enhanced cell response. In the current in vitro study, we assessed the effects of UV irradiation on the short-term (<45 min) interaction between hylan gels and human blood cells (RBCs, platelets) and coagulation proteins at physiologic temperature. Although the lowered hydrophilicity of irradiated (UV) hylans elicited greater vascular cell response relative to unmodified (U) hylans, platelet deposition was unaffected and much lower compared to collagen-coated glass controls. The adhered platelets were rounded or mildly pseudopodic and did not express p-selectin, an activation marker. Both gel types induced identical, and minimal platelet release as measured using an platelet factor 4 ELISA, and identically deferred the intrinsic and extrinsic coagulation pathways. Both gel types induced elevated levels of contact activation of bound, but not plasma-phase factor XII relative to controls. Hemolysis rates were also identical and within accepted standards. We conclude that UV-treatment of hylans, useful to improve surface endothelialization, does not compromise their short-term hemocompatibility, vital to their use as vascular implant materials.     

Immunohistochemical and molecular genetic study on epithelioid glioblastoma: Series of seven cases with review of literature

Epithelioid glioblastoma (e-gbm) is a recently described variant of glioblastoma (GBM) which is associated with short survival and now added as a provisional entity to WHO 2016 classification of CNStumors. About half of these tumors show characteristic BRAF-V600E mutation. However, unlike conventional GBMs, e-gbm lack specific diagnostic and prognostic markers. Hence, we aimed to molecularly characterize these tumors. An extensive review of literature was performed.In a multi-institutional effort, all the cases of glioblastoma of year 2017 were reviewed. Cases with predominant epithelioid morphology were analysed. Seven cases of e-gbm (adults:4 and pediatric: 3) were identified. Duration of symptoms varied from 2 weeks to one month. Radiologically, all cases were supratentorial, contrast enhancing with solid and cystic appearance. Majority of the cases were immunopositive for GFAP (71%), EMA (71%), S100 (71%) and vimentin (85%). All the cases showed ATRX, INI-1 and H3K27me3 expression. BRAFV600Emutation was seen in 28% of cases. TERT mutation was seen in 40% cases, while one case showed EGFR amplification. H3F3A mutations and PTEN deletions were seen in none. Although e-gbms are rare, epithelioid morphology of a CNS tumor in a young adult or children with areas of necrosis needs thorough histomorphological and genetic workup.     

Searching for the initiating site of the major capsid protein to generate virus-like particles for a novel laboratory mouse papillomavirus

Correctly folded virus-like particles (VLPs) of papillomavirus (PV) display conformationally dependent epitopes that are type specific, maintained on authentic virions, and induce neutralizing antibodies. Alignment of the L1 amino acid (aa) sequences of 84 PVs revealed that the lengths of their N-termini are diverse and that multiple, possible initiation methionine (met) codons exist. The L1 gene of MusPV (MmuPV1), that naturally infects immunodeficient laboratory mouse strain (NMRI-Foxn1^nu/Foxn1^nu), has four met codons at the 1st, 2nd, 28th, and 30th aas from its N-terminus. Of these, the 3rd and 4th mets, that are at the 28th and 30th aa position from the N-termius, respectively, are located at the position where most PVs have their first met. These two mets, located at the 9th and 11th from the YLPP conserved aas of most PVs, should be considered as consensus initiation codons of PV L1s. Three L1 proteins of MusPV, starting from the 2nd, 3rd, and 4th mets, were expressed using a baculovirus expression system and characterized for their ability to self-assemble into VLPs. While MusPV L1 proteins starting from the 2nd met expressed an L1 protein that did not fold into VLPs, the L1s starting from the 3rd and 4th mets generated correct VLPs in abundant quantities. We now conclude that the highest quantity and best quality VLPs are made from the consensus L1 met of MusPV.     

Cytopathology of uncommon malignant renal neoplasms in the pediatric age group

Malignant renal neoplasms are common solid tumors in pediatric oncology practice. These include the common Wilms' tumor/nephroblastoma and the uncommon neoplasms such as clear-cell sarcoma of the kidney (CCSK), rhabdoid tumor, renal-cell carcinoma, and others. The aim of this study was to describe in detail the cytopathological features of the histopathologically proven uncommon pediatric renal tumors. Aspirates from Wilms' tumor, which are mesenchyme predominant, show clusters of spindle cells associated with the matrix material. Evidence of rhabdomyoblastic differentiation may be present. CCSK, classic subtype, is characterized by round to oval cells arranged perivascularly and also in sheets and clusters intimately associated with a metachromatic matrix mucopolysaccharide material better appreciated in May-Grunwald-Giemsa (MGG)-stained smears. The cells also have more abundant cytoplasm and may show nuclear grooves. Spindle-cell pattern of CCSK is difficult to diagnose on aspiration cytology. Renal-cell carcinoma of childhood shows similar cytological features as its adult counterpart. Rhabdoid tumor of the kidney is characterized by a monomorphic population of cells with abundant cytoplasm, eccentric nuclei with prominent nucleoli. Intrarenal yolk sac tumor is a rare neoplasm and shows severely pleomorphic cells on aspiration.Awareness of these entities is important for the practicing cytopathologist. Further, non-Wilms' renal malignant neoplasms must be distinguished from the common Wilms' tumor so that appropriate chemotherapy protocols may be instituted in cases where the tumor is in an advanced stage of malignancy.     

Fine-needle aspiration in the evaluation of thyroid lesions in children

The role of fine-needle aspiration (FNA) cytology in the evaluation of thyroid lesions in not as well established in children when compared with adults. Hence we aimed to ascertain the utility and limitations of FNA in childhood thyroid lesions. This was a retrospective analysis of all thyroid FNA performed in children less than 14 years of age over a 4-year period (2005-2009). Histopathological follow-up was available in six cases. A total of 77 cases were included in the analysis. The most common cytological diagnosis was lymphocytic thyroiditis (49.3%), followed by colloid goiter (18.2%), hyperplasia (10.4%), and benign aspirate (7.8%); malignancy was identified in six cases (7.8%). Of these six cases, three were papillary thyroid carcinoma. There was one false-positive case reported as a Hurthle-cell neoplasm, which on histology showed Hashimoto's thyroiditis. One case each of rhabdomyosarcoma and spindle epithelial tumor with thymus like differentiation was wrongly diagnosed as thyroid neoplasm, NOS, and medullary carcinoma (spindle variant), respectively. The overall diagnostic accuracy was 98.6% with 100% sensitivity, 98.6% specificity, 80% positive predictive value, and 100% negative predictive value. FNA is extremely valuable in the initial evaluation of thyroid swelling in children. Rare neoplasms masquerading as thyroid nodules in children can pose difficulties in diagnosis; however, papillary carcinoma is easily recognized. In lymphocytic thyroiditis, it provides a tissue diagnosis, thereby avoiding more invasive procedure for merely diagnostic purposes.