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Identification and classification of viruses that have not been propagated.

Microorganisms that cannot be grown in the laboratory can now be tentatively identified, by cloning and sequencing particular nucleic acid segments and then carrying out a comparative sequence analysis with an appropriate database. For bacteria, a few universally distributed genes and gene products have enabled comparative sequence analysis to be used for tentative identification and classification of uncultured bacteria. For viruses, there is no universally distributed viral gene or gene product. However, in a few cases, viruses that could not be propagated in the laboratory have been identified and classified. In these cases, either the entire viral genome sequence was determined or partial sequence information was supplemented with additional data. The Executive Committee of the International Committee on Taxonomy of Viruses (ICTV) has reviewed the issue of identification and classification of viruses that have not been propagated. Under the ICTV system, formal review of any taxonomic proposal is carried out by the relevant ICTV Subcommittee or Study Group. The few examples of unpropagated viruses that have arisen thus far have been readily accommodated within existing viral taxonomy, with the international group of experts comprising each Subcommittee and Study Group determining the necessary and sufficient amount of information needed for classification of an unpropagated virus on a case-by-case basis.

Bacteria↗

Increased detection and yield of adenovirus in caffeine-stabilised KB cell monolayers.

Viable monolayers of KB cells were maintained without passage for up to one year by adding 2 X 10(-3) M caffeine to the medium, while untreated monolayers degenerated after 2 weeks. When adenoviruses type 2 and 17 were titrated on caffeine-stabilised KB cells, the late breakthrough of cytopathic effect at terminal dilutions (up to 40 days after inoculation) resulted in titre determinations up to 100-fold higher than those measured before spontaneous degeneration in untreated cells. Yields of adenovirus type 2 in caffeine-treated KB cultures infected at different multiplicities and harvested at different intervals were equal to, or up to 100-fold higher than those obtained in untreated cultures.

Adenoviruses, Human↗

Characterization of sublines of Epstein-Barr virus producing HR-1 cells and its implication in virus propagation in culture.

To understand the mechanism regulating the EBV replication cycle, several sublines were obtained from HR-1 cells by the limiting dilution method. Based on their biochemical and molecular characteristics, these sublines can be categorized into two classes: the high EBV-DNA containing (H) subline and low EBV-DNA containing (L) subline. The amount of EBV proteins, such as EBV polymerases, EBV DNase, EAD, ZEBRA, MA, and VCA, was much higher in H sublines than in L sublines. Only 20% of cells in the H subline express those proteins. In addition to regular EBV DNA restriction enzyme fragments, additional DNA restriction enzyme fragments, as detected by different EBV DNA fragment probes, were found to be present in H sublines but not in L sublines. No BamH1 W-Z DNA fragment rearrangement, which was the primary reason for ZEBRA expression in a high EBV-DNA containing subline, Clone 5, was found in H sublines. When L sublines were treated with 12-0-tetradecanoylphorbol-13-acetate and sodium butyrate, EBV-specific proteins, including ZEBRA protein, could be induced in cells, but no virus could be detected in the medium. Thus, the lack of EBV production by L sublines is more than the simple lack of expression of ZEBRA protein. L sublines are susceptible to EBV infection and are capable of producing EBV after infection. The importance of the presence of L cells in the H subline for the propagation of EBV in culture is suggested.

Burkitt Lymphoma↗

Increased production of human immunodeficiency virus (HIV) in HIV-induced syncytia formation: an efficient infection process.

Syncytia or multinucleated giant-cell formation is one of the major cytopathic effects induced by human immunodeficiency virus (HIV) infection. Cell fusion results from the strong interaction of CD4 molecules on the surface of the uninfected T cells and gp120, an external envelope glycoprotein of HIV on the infected T cells. We studied the production of HIV in fusion cells between MOLT-4 and virus-infected MOLT-4/HIV cells and found that HIV production was enhanced up to three- to fivefold, which showed a good correlation with the appearance and extent of syncytia formation. Blocking the fusion by monoclonal antibody against a binding epitope of CD4 molecule to gp120 decreased the HIV production significantly. Enhancement of HIV production was observed by more than five-fold in comparison with chronically infected cells, which were fusion free 20 hr postcocultivation. Electron microscopic observation also showed the presence of abundant HIV particles inside the fused cells and on the outer surface. AZT blocked the HIV augmentation of fused cells in coculture completely. Southern blot analysis revealed that both integrated and unintegrated HIV DNA were highly accumulated in fusion cells, as compared with fusion-free MOLT-4/HIV cells. Among unintegrated DNA, circular and linear DNA were accumulated to a similar degree. Northern blot hybridization showed that rapid enhancement of all three species of HIV-specific RNA containing genomic (9.2 kb) and subgenomic (4.3 and 1.9 kb) RNAs were found 20 hr postinfection in fusion cells. These data suggest that syncytia formation is an extremely active infection process of HIV, by which multiple rounds of reinfection might take place.

Animals↗

Susceptibility of chick neural retina to viral multiplication in vitro during embryonic development.

Decrease in the susceptibility of embryonic chick neural retina cultures to the multiplication of various viruses was observed with increasing age of the embryo. In contrast the retinal cells supported the multiplication of Sindbis virus irrespective of the age when they were infected with the viral RNA. These results suggest that the restricted multiplication of the viruses observed is due to the modulated inability of the cell to process the adsorbed viruses for subsequent replication.

Aging↗

Isolation and some serological and epidemiological data on the viruses recovered from patients with subacute thyroiditis de Quervain.

Virological and serological methods were used in examination of 28 patients suffering from subacute thyroiditis de Quervain. Attempts to isolate a presumed viral agent from 8 patients were performed by inoculation of serum, urine, and aspiration biopsies of thyroid glands taken at different stages of the illness, into tissue cultures of different types of human and animal cells. Recovery of a cytopathic viral agent on cells of a rabbit lung continuous line was successful in 5 cases. Serological cross reactions exist between the isolated viruses and patient serum but not with serum of healthy people. Cases with the acquired illness and positive antibodies against the isolated viruses who had been in close and prolonged contact with patients suffering from subacute thyroiditis de Quervain were also investigated.

Acute Disease↗

The influence of iododeoxyuridine pretreatment of various cell cultures on adenovirus multiplication.

Treatment of cell cultures with iododeoxyuridine (IUdR) before virus inoculation may enhance the subsequent virus multiplication. This effect was studied in seven kinds of cell cultures with seven human adenoviruses from six subgenera. IUdR (50 micrograms/ml) was added 24 h after the seeding of cells, left for 2 days, and removed before virus inoculation. IUdR had no effect on cell proliferation. Viral cytopathic effect was enhanced in many instances by IUdR pretreatment. However, virus multiplication was enhanced weakly in only four cases [adenovirus 7 in Vero, African green monkey kidney, cynomolgus monkey kidney; adenovirus 4 (Ad4) in HeLa cells], and strongly only for Ad4 in Vero cells, which are semipermissive for Ad4. IUdR pretreatment of Vero cells shortened the replication cycle for Ad4 and increased sensitivity for minimal virus concentrations about 1000-fold. From immunofluorescence experiments it appears that more than one infectious particle is required to infect an untreated Vero cell.

Adenoviridae↗

Isolation of herpes simplex virus clones and drug resistant mutants in microcultures.

Suitable dilutions of herpes simplex virus (HSV) preparations inoculated into microcultures of confluent monolayers of human foreskin or Vero cells, in individual wells of plastic "microplates", induced viral cytopathic effects that resulted from the infection of the cultures by single virus particles. The clonal nature of the viral progeny in isolated wells was supported by visual control over the development of viral foci and by statistical analysis. The method has the advantage of speed and economy, while it also yields a large primary clonal virus stock. HSV clones resistant to phosphonoacetic acid (PAA) and 5-iodo-2'-deoxyuridine (IUdR) could be readily isolated by the described technique.

Adsorption↗

Attempts to establish the scrapie agent in cell lines.

Attempts were made to establish a persistent infection with scrapie agent in four murine cell lines. Of the four lines tested, viz. P388D1, P3-NS1-Ag-1 (NS1), L cells and an NS1 spleen cell hybrid, only the NS1 cell line showed any evidence of agent replication. Ten per cent dimethylsulphoxide (DMSO) was included in the culture media of L cells inoculated with scrapie agent. This treatment raised the initial levels of scrapie agent associating with the L cells but did not result in a persistently infected cell line. An effect of DMSO in the inoculum was observed in mice inoculated intraperitoneally, the incubation period of the disease was considerably shortened.

Animals↗