CYTOPATHOGENICITY OF INFLUENZA VIRUS (H4 N3)

Monolayer tissue cultures of chicken embryo fibroblast ( CEF ) cells infected with avian influenza virus isolate were examined by the hematoxylie and eosin (H&E) staining and indirect immunoperoxidase test for studying the cytopathogenic effect of the virus. Cytopathological changes which occurred in the uncleus of infected cells included nuclear and nucleolar hypertropy, chromatin margination and intranuclear inclusions. The most striking cytoplasmic change were the presence of perinuclear. eosinophilic inclusions at 22-36 hours post inoculation ( p. i.). Vacuolization, and granulation were also observed. Indirect immunoperoxidase ( IP ) test demonstrated the localization of influenza virus antigens in infected cells. A positive peroxidase reaction observed in the nucleus and cytoplasm were similar to those shown hematoxyline and eosin staining.

A Cytopathic Effect-Based Tissue Culture Method for HCoV-OC43 Titration Using TMPRSS2-Expressing VeroE6 Cells

ABSTRACT Human coronavirus (HCoV)-OC43 rarely shows a cytopathic effect (CPE) after infection of various cell lines, and the indirect immunoperoxidase assay (IPA), a relatively complex procedure, has long been used as an alternative assay. Because HCoV-OC43 uses cell-surface transmembrane protease serine 2 (TMPRSS2) for cell entry, VeroE6 cells expressing TMPRSS2 may show a clear CPE after HCoV-OC43 infection. The aim of this study was to construct a 50% tissue culture infectious dose (TCID50) assay for HCoV-OC43 based on CPE evaluation using VeroE6/TMPRSS2 cells. VeroE6/TMPRSS2 cells showed clear CPEs 3 to 4 days after low-titer HCoV-OC43 infection. Evaluation of viral kinetics indicated that the viral titer in the culture supernatant of VeroE6/TMPRSS2 cells in the early stages of infection was higher than that of other cells. In comparison, between the CPE-based and the IPA-based (i.e., the reference titer) methods, the titer measured with CPE evaluation 4 to 5 days after infection using VeroE6/TMPRSS2 cells showed a much smaller difference from the reference titer than that measured using other cells. Thus, the TCID50 assay using CPE evaluation with VeroE6/TMPRSS2 cells provides the correct titer value and will greatly contribute to future research on HCoV-OC43. IMPORTANCE HCoV-OC43 rarely shows a cytopathic effect (CPE) in infected cell lines, and thus the plaque and TCID50 assays by CPE observation are not applicable for titration; the indirect immunoperoxidase assay (IPA) is used instead. However, the IPA is relatively complex, time-consuming, costly, and not suitable for simultaneous titration of many samples. We developed a TCID50 assay using CPE evaluation with TMPRSS2-expressing VeroE6/TMPRSS2 cells that provides the same accuracy as the conventional IPA-based viral titration and does not require any staining procedures using antibodies or substrates. This titration method will greatly contribute to future research on HCoV-OC43 by allowing simple, low-cost, and accurate titration of this virus.

Factors Affecting The Indirect Immunoperoxidase Staining Of Infectious Bursal Disease Virus Antigens In Cells

THE application of the Immunoperoxidase (IP) antibody technique for the definitive diagnosis of Infectious Bursal Disease (IBD) in chickens has earlier been described (Okeke and Lang, 1982). During that study it was noted that the Indirect method of IP was more sensitive in the demonstration of IBD viral antigens than the direct method. Consequently, more formation was sought on the specific technical parameter and the consequences that resulted by deviations from the codified procedures employed in the Indirect technique with the aim of obtaining an even better result. Studies of pH, serum dilutions, serum incubation time, conjugate dilutions, conjugate incubation time and developing time were conducted to find out what Influence such factors could have on the final staining product.

C-fos, ERK1/2, MAP2, NOTCH1 Proteins Expression Patterns in Human Cerebral Cortex Neurons after Ischemic Stroke

Background. The search for protein (these include c-fos, ERK1/2, MAP2, NOTCH1) expression that provide neuroplasticity mechanisms of the cerebral cortex after ischemic stroke (IS) patterns is an urgent task. Aims to reveal c-fos, ERK1/2, MAP2, NOTCH1 proteins expression patterns in human cerebral cortex neurons after IS. Materials and methods. We studied 9 left middle cerebral artery (LMCA) IS patients cerebral cortex samples from 3 zones: 1 the zone adjacent to the necrotic tissue focus; 2 zone remote from the previous one by 47 cm; 3 zone of the contralateral hemisphere, symmetric to the IS focus. Control samples were obtained from 3 accident died people. Identification of targeted proteins NSE, c-fos, ERK1/2, MAP2, NOTCH1 was performed by indirect immunoperoxidase immunohistochemical method. Results. Moving away from the ischemic focus, there is an increase in the density of neurons and a decrease in the damaged neurons proportion, the largest share of c-fos protein positive neurons in zone 2, NOTCH1 positive neurons in zone 1, smaller fractions of ERK1/2 and MAP2 positive neurons compared to the control only in samples of zone 1. Conclusions. With the IS development, the contralateral hemisphere is intact tissue increased activation zone, while the zones 1 and 2 have pathological activation signs. In zone 1 of the range, the adaptive response of the tissue decreases, and in zone 2 it expands. Therefore, a key target for therapeutic intervention is zone 2.

Conventional and Molecular Detection of Avipoxviruses from Chickens, Pigeons and Turkeys

In the present study, a total of 90 cutaneous lesions samples were collected from chickens, pigeons, and turkeys farms in Dakahlia Governorate, Egypt during summer 2016. These farms suspected to be infected with Avipoxviruses (APVs).Thirty pooled samples were created (10 from chickens, 10 from pigeons and 10 from turkeys). Hyperimmune serum was prepared against standard fowlpox virus in adult white New Zealand rabbits. APV were identified in the collected samples using agar gel precipitation test (AGPT), indirect immunoperoxidase, and polymerase chain reaction (PCR) based on 4b gene of APVs. The results revealed that out of 30 tested samples there were 16 samples (53.3%) tested positive via AGPT including, 6 chicken samples (60%) , 5 pigeon samples (50%) and 5 turkey samples (50%). while using indirect immunoperoxidase, positive results were detected in 23 samples (76.7%) including, 8 chicken samples (80%), 8 pigeon samples (80%) and 7 turkey samples (70%).The 4b gene of APVs was detected using PCR in all tested samples (100%). In conclusion, Indirect immunoperoxidase is superior over AGPT in APVs detection in collected samples from chickens, pigeons and turkeys. PCR could be efficiently used in molecular diagnosis of the virus.

NO-tryptophan: a new small molecule located in the rat brain

<p>A highly specific monoclonal antibody directed against nitric oxide-tryptophan (NO-W) with good affinity (10^-9M) and specificity was developed. In the rat brain, using an indirect immunoperoxidase technique, cell bodies containing NO-W were exclusively found in the intermediate and dorsal parts of the lateral septal nucleus. No immunoreactive fibres were found in the rat brain. This work reports the first visualization and the morphological characteristics of cell bodies containing NO-W in the mammalian brain. The restricted distribution of NO-W in the rat brain suggests that this molecule could be involved in specific physiological mechanisms. </p>

Inhibition ofPasteurella multocidaAdhesion to Rabbit Respiratory Epithelium Using Lectins

This study aimed to evaluate the ability of a panel of lectins to inhibit the ability ofPasteurella multocidato adhere to and affect the rabbit respiratory epithelium. Nasal septa from rabbit fetuses were cultured with various lectins before the addition ofP. multocida. The percentage of bacteria adhering to the epithelium was evaluated semiquantitatively by indirect immunoperoxidase (IIP) staining. The goblet cells (GCs) were counted in semithin sections stained with toluidine blue and served as the main morphological criterion to evaluate the inhibitory effect of the lectins. The lectins PNA, WGA, RCA120, and DBA significantly inhibited the adhesion ofP. multocidato the ciliated epitheliumP<0.05and prevented the pathogen-induced increase in the number of GCsP<0.05compared with those of positive control tissues. In addition, VVA, SJA, UEA I, DSL, SBA, and ECL significantly inhibited the increase in GCs compared with that of the control tissues. The results suggest that less aggressive therapeutic strategies, such as treatment with lectins, may represent alternative approaches to control bacterial respiratory infections.

Isolation of Infectious Bronchitis Virus in Primary cells of the Chick Embryo Chorioallantoic Membrane

The susceptibility of the primary chick embryo chorioallontoic membrane cells to infectious bronchitis virus was evaluated after twenty consecutive passages in chick embryo chorioallontoic membrane cells. Virus replication was monitored by cytopathic observation, indirect immunoperoxidase, and reverse transcription polymerase chain reaction (RT-PCR). At 72 hours post-infection in third passage, the cytopathic effect was characterized by rounding up of cells, monolayer detachment, intracytoplasmic brownish colouration was readily observed by immunoperoxidase from 24hours p.i in third passage, and at all times the extracted viral RNA from IBV-infected monolayers was demonstrated by RT-PCR. Tissue culture ineffective dose50 (TCID50) was used to measure virus titration performed on primary chick embryo chorioallontoic membrane cells and the titre in twenty passage was 108.6 TCID50/ml. The results obtained in this study suggested that the primary chick embryo chorioallontoic membrane cells can be used for adaptation infectious bronchitis virus (IBV) and may be considered a step forward for the use of these cells in the future for IBV vaccine production