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At least 73 records · Page 4Linked to original sources
PubMed2000

Nested PCR for rapid detection of mumps virus in cerebrospinal fluid from patients with neurological diseases.

In this study, we have developed a reverse transcription (RT)-nested polymerase chain reaction (n-PCR) for the detection of mumps virus RNA in cerebrospinal fluid (CSF) from patients with neurological infections. A specific 112-bp fragment was amplified by this method with primers from the nucleoprotein of the mumps virus genome. The mumps virus RT-n-PCR was capable of detecting 0.001 PFU/ml and 0.005 50% tissue culture infective dose/ml. This method was found to be specific, since no PCR product was detected in each of the CSF samples from patients with proven non-mumps virus-related meningitis or encephalitis. Mumps virus RNA was detected in all 18 CSF samples confirmed by culture to be infected with mumps virus. Positive PCR results were obtained for the CSF of 26 of 28 patients that were positive for signs of mumps virus infection (i.e., cultivable virus from urine or oropharyngeal samples or positivity for anti-mumps virus immunoglobulin M) but without cultivable virus in their CSF. Overall, mumps virus RNA was detected in CSF of 96% of the patients with a clinical diagnosis of viral central nervous system (CNS) disease and confirmed mumps virus infection, while mumps virus was isolated in CSF of only 39% of the patients. Furthermore, in a retrospective study, we were able to detect mumps virus RNA in 25 of 55 (46%) CSF samples from patients with a clinical diagnosis of viral CNS disease and negative laboratory evidence of viral infection including mumps virus infection. The 25 patients represent 12% of the 236 patients who had a clinical diagnosis of viral CNS infections and whose CSF was examined at our laboratory for a 2-year period. The findings confirm the importance of mumps virus as a causative agent of CNS infections in countries with low vaccine coverage rates. In summary, our study demonstrates the usefulness of the mumps virus RT-n-PCR for the diagnosis of mumps virus CNS disease and suggests that this assay may soon become the "gold standard" test for the diagnosis of mumps virus CNS infection.

Central Nervous System Diseases↗
PubMedDate not supplied

Use of polyethyleneglycol-treated serum for animal cell cultures.

Although many proteins will be removed from sera by precipitation with 10% polyethyleneglycol (PEG) the growth-promoting properties of such PEG-treated sera for many cell lines and cell strains are hardly reduced. Among the precipitated proteins are the macroglobulins which are difficult to remove from cell cultures and which may cause allergic reactions if incorporated into vaccines. The gammaglobulins are removed in this way as well. If the sera are contaminated with viruses or phages the titres will be reduced by approximately 4 logs and thus the incidence of virus contamination will be reduced by the PEG-treatment. For foot-and-mouth Disease (FMD) vaccine production PEG-treated sera from vaccinated cattle were applied for cell and virus cultivation. The virus obtained was subsequently precipitated with PEG and collected by filter-aid filtration. A concentrated virus product was obtained, which was practically devoid of serum proteins.

Animals↗
PubMed1997

Immunohistochemical detection of VHS virus in paraffin-embedded specimens of rainbow trout (Oncorhynchus mykiss): the influence of primary antibody, fixative, and antigen unmasking on method sensitivity.

The influence of the primary antibody, the fixative, and the antigen unmasking technique on the method sensitivity of immunohistochemistry as a method for the identification of viral hemorrhagic septicemia (VHS) virus in paraffin-embedded specimens of naturally infected rainbow trout (Oncorhynchus mykiss) was examined. Fish (200-300 g) were collected during an outbreak of VHS. Parallel specimens from liver, spleen, kidney, and brain were fixed by immersion in 10% phosphate-buffered formalin, periodate-lysine-paraformaldehyde (PLP), Bouin's fluid, or absolute ethanol. Virus cultivation was also performed on parallel specimens, and the virus titer (TCID50/ml) was determined. Purified nucleocapsid protein (N-protein) of the virus was incorporated in an artificial antigen substrate polymerized bovine serum albumin), fixed as described above, and embedded in paraffin wax. Microwave unmasking was performed on formalin-, PLP-, and Bouin's fluid-fixed specimens. The presence of virus peptides in situ or N-protein in the artificial antigen substrates was visualized using an immunohistochemical method based on alkaline phosphatase or peroxidase and one polyclonal and five monoclonal polypeptide-specific antibodies. VHS virus was identified in situ in specimens with high virus titers (10(7-8) TCID50/ml) regardless of the fixative and without the need of an unmasking procedure. A pronounced masking effect was observed for the cross-linking formalin and PLP fixatives. Regardless of the primary antibodies used, there was a significantly higher epidemiologic sensitivity (the proportion of virus positive samples that tested positive by immunohistochemistry) using ethanol and Bouin's fluid compared with formalin and PLP (P < 0.05). At 10(5) TCID50/ml, the average sensitivity reached 0.5, and at > or = 10(6) TCID50/ml, sensitivity was 0.9. Unmasking procedures showed a moderate effect and did not result in significantly higher epidemiologic sensitivity (P = 0.17), There was great variation for the different monoclonal antibodies/antigens and fixatives. Sensitivity studies on antigen substrates were in accordance with results of in situ studies that showed the highest sensitivity for ethanol and Bouin's fluid. Virus cultivation was more sensitive than immunohistochemistry. This study showed that the fixative and the primary antibody both influence method sensitivity and that VHS virus antigens concealed during fixation are difficult to reexpose. Immunostaining for VHS virus should be performed with monoclonal antibodies specific for the N-protein, and tissue samples should be fixed in either ethanol or Bouin's fluid. Immunohistochemistry is specific but is less sensitive than virus cultivation. Immunostaining for VHS virus can be a valuable supplement to virus cultivation during acute outbreaks of disease.

Animals↗
PubMed1976

Electron microscopic studies of viral agents in Crohn's disease.

Viral agents isolated from ileal filtrates of patients with Crohn's disease have been successfully cultivated in continuous rabbit ileum tissue-culture and grown to titres sufficient to allow electron microscopic characterisation. Electron microscopic studies showed clusters of viral particles only in tissue-cultures inoculated with cultivated viruses originally obtained from tissues from patients with Crohn's disease. Control cultures showed no evidence of viral agents. The mean virus-particle diameter was 30 nm. An electron-dense central core was present and a spiculated coat was demonstrated. The physical chemical properties of this agent, as previously described, together with the electron microscopic appearance are consistent with the appearance of a picornavirus.

Cell Line↗
PubMedDate not supplied

[The isolation of a concentrated and purified rotavirus preparation by using bentonite].

The preparation obtained when cultivating virus SA-11 in the transplantable cell culture SPEV has been purified and concentrated by means of bentonite. The virus adsorption at acidic values of pH (4.0-4.5) and elution at alkaline values of pH (8.5) were the main stages of production of the concentrated and purified rotavirus preparation. The paper embraces data on the infective activity of the virus protein content, amount of the rotavirus antigen, results of the electron and immunoelectron microscopy, characterizing the method efficiency.

Animals↗
PubMedDate not supplied

[Cultivation of the vaccine strain of the virus of porcine classic plague].

Several cell cultures were compared and examined for their capability to support reproduction of Classical Swine Fever Virus (CSFV). Experimental conditions of virus cultivation were optimized. The subculture of primary lamb testicular (LT) cells was selected for large cell cultivation of CSFV. In contrast to many cell lines, these cells were free of Bovine Viral Diarrhea Virus (BVDV) contamination partly due to liquid nitrogen storage and gamma-ray sterilization of serum used in the growth media. The vaccine strain CS of CSFV was obtained in the titers of 6.7-7.7 TCID50/ml under the optimal conditions of virus reproduction in the LT cells.

Animals↗