Persistently infected cultures as a source of hepatitis A virus

Primary African green monkey kidney, continuous African green monkey kidney cell line BS-C-1, and buffalo green monkey kidney cultures were infected with a uniform inoculum of hepatitis A virus (HAV). Although both the cell line BS-C-1 and primary African green monkey kidney cultures produced useful amounts of virus, HAV was detected earlier and in greater quantities in primary African green monkey kidney cultures. A persistently infected primary African green monkey kidney culture was developed. The influence of incubation time (4 to 40 days) and concentration (2 to 15%) of fetal calf serum in the maintenance medium on production of HAV by this culture was examined. An incubation period of 24 to 28 days was found to be optimal; reducing this period led to decreased yields of HAV. No significant difference in the amount of HAV produced was observed with differing concentrations of fetal calf serum. Three different methods of extraction and the effect of multiple extractions on the recovery of HAV from cell lysates were examined. Sonication was a critical factor. Two extractions yielded more than 90% recoverable virus. Yields in excess of 10(11) physical particles of HAV per 850-cm2 roller bottle were routine. The total yield could be increased by concentrating the HAV present in spent maintenance medium by using bentonite or organic flocculation.     

Characterization of long-term cultures of hepatitis C virus.

The human T- and B-cell lines HPBMa10-2 and Daudi produced infectious hepatitis C virus (HCV) for more than 1 year after infection. The infectivity titer of the cell culture-grown HCV and its genome titer were comparable. The virion density in sucrose was around 1.12 g/ml. Among the 13 variants detected in the inoculum, 7 were adsorbed by the cells and one particular HCV sequence which was present in minor quantities in the inoculum persisted.     

Global market: shellfish imports as a source of reemerging food-borne hepatitis A virus infections in Spain

A total of 16 mollusk imports from South America to Spain, including clam and scallop species, were analyzed for hepatitis A virus (HAV), due to the great concern about this type of food after an important hepatitis A outbreak in eastern Spain in September 1999. In addition, clams from the stock that had caused the outbreak were also tested. Of the 17 stocks, four were positive for the presence of HAV RNA as demonstrated by RT-PCR and Southern hybridization. Contradictory analyses confirmed the results of the primary tests in all cases. The findings obtained in this work strongly support the role of mollusk imports from endemic areas of HAV as an important vehicle of hepatitis A, and demonstrate the imperative need for sanitary control measures to prevent future outbreaks of this disease. Electronic Publication     

Animal cell cultures as a source of hormones

Current methodology now makes it possible to establish in culture a variety of mammalian cells which perform organ-specific functions during serial propagation for periods of months or years. This report describes the results of experiments with animal and human cells that produce growth hormone, adrenocortical steroid hormones, thyrocalcitonin, and parathyroid hormone. Within the next decade it should be possible to use cell culture methods for manufacturing purposes to produce hormones and other valuable cellular products which are difficult to obtain in other ways. At first it may be necessary to use neoplastic cells for this purpose; but evidence is accumulating to suggest that it may eventually be possible to establish in culture normal, functional animal and human cells.     

Evidence of hepatitis E virus replication on cell cultures.

Several human and animal cell lines have been used to grow hepatitis E virus. The strain SAR-55 was adapted only on PLF/PLC/5 cell line without any visible cytopathic effect. The growth of the SAR-55 was monitored by examining the positive and the negative strands of HEV-RNA. Stool samples, obtained from hospitalised acute hepatitis patients at the Fever Hospital of Alexandria (Egypt), were used to confirm the susceptibility of PLF/PLC/5 cells. After more than one-week's cultivation, three stool samples out of 17 IgM anti-HEV positive and 1 from 52 IgG anti-HEV positive patients showed a specific RT-PCR amplification product. The nucleotide sequences of the methyltransferase region of the genome in the isolates revealed the maximum homology with Burma strain with several point mutations.     

A filterable virus as a causative agent of epidemic hepatitis

Summary In four patients suffering from epidemic hepatitis we succeeded in isolating from the blood during the fever period and from the urine during the jaundice, a filterable virus, which is pathogenic to the guinea pig and which can be inoculated to this animal in different ways, but by preference intraperitoneally. Fever, which sometimes lasts only one day, is the only morbid symptom observed in guinea pigs.In these animals the virus can be shown in the organs and in the blood during the fever period; after that it is excreted with the urine during apparently a short period. The virus has been grown on the chorioallantois of the chick embryo so far during 20 passages. The virus is resistant to glycerol, drying and low temperatures, not to formaline and heating.In the livers of the guinea pigs focal changes (degeneration, dissociation, necrobiosis, yellow liver-atrophy) may be found. After dulling through the disease immunity occurs, whilst in the serum of recovered patients and guinea pigs neutralizing antibodies can be detected.     

Dried blood spots as a practical and inexpensive source for human immunodeficiency virus and hepatitis C virus surveillance

Passive surveillance of infectious diseases with a high percentage of asymptomatic cases or long incubation periods, such as acquired immunodeficiency syndrome (AIDS), does not reflect the current transmission dynamics. Thus, a multi-strategic surveillance, such as the human immunodeficiency virus (HIV) sentinel surveillance proposed by the World Health Organization (WHO), is necessary. The Brazilian HIV sentinel surveillance was started in May 1992 with this purpose. The objectives of this study were to evaluate the feasibility and costs of HIV and hepatitis C virus (HCV) surveillance using dried blood spots (DBS) collected for neonatal screening of metabolic diseases in the state of Minas Gerais, Brazil. This was accomplished through the comparison of HIV and HCV seroprevalence with previous Brazilian studies. From December 2001 to June 2002, 24,905 newborns were tested for HIV and 4211 for HCV. HIV seroprevalence was 0.25% and the 95% confidence interval (CI) was 0.18, 0.31%; and HCV seroprevalence was 0.71% and the 95% CI was 0.46, 0.97%. These numbers are similar to previous Brazilian studies. Cost in this study was approximately USD 3.10 per sample, which was roughly one third of the cost of the same exam at the Brazilian HIV sentinel surveillance. We conclude that it is possible and more cost-effective to use DBS for infectious diseases surveillance, albeit it is still necessary to compare these results with the usual sentinel methodology in a concomitant trial.     

Growth of organ cultures of mouse liver infected with Coxsackie A13 virus

Peculiarities attending the growth and proliferation of the organ cultures of the liver of mongrel albino mice infected once with Coxsackie A-13 virus were investigated. A marked zone of growth, mostly of the epithelial cells, was determined rather early in the liver explants of mice in the experimental group, whereas in control group of mice the cell growth around the explant of the liver was either absent or very weak. Besides, a great number of lymphocytes evenly arranged in the zone of hepatocytes growth was observed in the preparations of the experimental mice liver. Lymphocyte "adhesion" to hepatocytes of the culture was revealed in some preparations. Moreover, destruction of the hepatocytes and a marked rarefaction of the cell layer occurred at the sites of lymphocytes accumulation on the 21st and the 28th days of growth.     

Clonal analysis of mammalian cell cultures persistently infected with Japanese encephalitis virus.

More than 200 cells were cloned from populations of mammalian cells persistently infected with Japanese encephalitis virus. Only four cloned cultures contained cells that had viral antigen measurable by immunofluorescence and that released infectious virus, yet all clones harbored virus-specific RNA. Superinfection of cloned cells with wild-type Japanese encephalitis virus did not produce cytopathic effects, but resulted in production of viral antigen and infectious virus in formerly nonproducing clones. Cocultivation of nonproducer clone cells with normally permissive cells did not induce virus production, nor did treatment of nonproducer clones with various inhibitors of DNA, RNA, or protein synthesis. It is suggested that the cloning procedure may have selected for a particular subpopulation of cells and that defective virus is also involved in establishment and maintenance of persistent infection.     

Skin explant cultures as a source of keratinocytes for cultivation

Cultivated human keratinocytes can be used successfully in the treatment of burn patients, but efforts to heal burns and other wounds can be hampered by the very small skin biopsies available for cultivation of transplantable keratinocyte sheets. A small biopsy (and correspondingly small number of enzymatically isolated keratinocytes for use in classical cultivation techniques) can lead to a low yield of multilayer sheets for clinical application or unacceptably long cultivation times. One way of addressing this is to make use of skin remnants remaining after enzymatic digestion and culture cells migrating out of these skin explants. Sufficient numbers of explant-derived keratinocytes can be obtained to facilitate additional routine cultivation of these cells. Biopsy remnants can be used to initiate explant cultures repeatedly (we were able to re-use pieces of skin 10 times and still obtain useful numbers of keratinocytes) and this “passaging” yields substantially more cells for classical cultivation than would be available from conventional methodology alone, and in a comparable timeframe. Another advantage of this method is that it does not require additional biopsies to be procured from already-compromised patients and overcomes problems associated with contamination of skin samples with resistant hospital-acquired bacterial infections common during prolonged hospitalization.