RESUMO
BACKGROUND: Rabies is a major zoonotic viral disease and is detected using the World Health Organization standard diagnostic techniques. Rabies detection is preferably done using the fluorescent antibody technique (FAT) that provides reliable diagnosis with almost 100% accuracy for all variant strains, if a proper conjugate is used. Rabies virus nucleoprotein (NP) is the most important protein used in production of a specific diagnostic conjugate. OBJECTIVES: The aim of this study was to extract the cell-associated rabies virus NP from infected Baby Hamster Kidney cell clone (BSR) with rabies virus (Pasteur vaccine strain/PV) and purify for a future project to produce an anti-NP conjugate. MATERIALS AND METHODS: Pasteur vaccine strain (PV) as the standard rabies vaccine strain with a focus-forming dose (FFD) of 105 was inoculated in to the BSR cell culture at a concentration of 10(6) cells per milliliter. Infected cells were harvested 72 hours after infection and the rabies NP was extracted from these cells by low-speed centrifugation and purification by ultracentrifugation in cesium chloride (CsCl) gradient. For analysis, the purified NP was subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). RESULTS: The volume of the lysate was 15 mL and it became 2.5 mL after purification, with a concentration of 3.25 mg/mL. The corresponding band to the cell lysate protein on the SDS-PAGE had a molecular weight of 50 KDa, similar to the molecular weight of NP in rabies virus. CONCLUSIONS: The rabies virus NP could be extracted and purified in an appropriate amount from infected cell culture. The results of SDS-PAGE analysis showed that the intact rabies virus NP had been purified properly and thus could be used for further steps to produce the specific diagnostic rabies conjugate.
RESUMO
BACKGROUND: Human enterovirus 71 (HEV71) was isolated for the first time from an infant with encephalitis in California in 1969 and then spread through the world. It has emerged as a major cause of a vast variety of diseases such as epidemics of hand, foot and mouth disease (HFMD), aseptic meningitis (AM), acute flaccid paralysis, and encephalitis. The aim of this study was to determine the prevalence of enterovirus 71 in children < 8 years old who were hospitalized due to primary diagnosis of AM in Tehran. METHODS: One hundred cerebrospinal fluid samples (CSF) were collected by physicians from children with a diagnosis of AM and transported on ice to the Pasteur Institute of Iran for further processing. Viral RNA was extracted and EV71 infection was detected by RT-PCR method using the specific primers. RESULTS: EV71 infection was detected in 14 patients (14%). Eight (57.14%) patients were younger than 2 years old, 11 (78.57%) were male and 3 (21.43%) were female. The seasonal peaks of EV71 were observed during autumn and winter with 6 (42.86%) and 5 (35.71%) cases, respectively. CONCLUSIONS: EV71 should be considered as a causative agent of AM in Iran with the epidemiological pattern similar to that of other enteroviruses as males are more susceptible to be affected by these viruses. Further studies on this virus are needed to improve our knowledge about them in our country.
