Science Vol 224, p 497-500 published 4 May 1984 (POPOVIC M. et al) "Detection, Isolation and Continuous Production of Cytopathic Retroviruses (HTLV-III) from patients with AIDS and Pre-AIDS".
MEDLINE (NLM Database) Accession number 85258784 Jpn. J. Cancer. (GANN) 1985, June 76 (6): 432-5 (Harada S., Yamamoto N) "Quantitative Analysis of AIDS-Related Virus Carrying Cells by Plaque-forming Assy Using an HTLV-I-positive MT-4 Cell Line".
Nature Vol 312, p 763-67 published 20/27 December 1984 (DALGLEISH A.G. et al) "The CD4 (T4) Antigen in an Essential Component of the Receptor for the AIDS Retrovirus".
| 1. | A method of detecting infectious AIDS virus in a fluid sample, such as blood serum or other tissue fluids, wherein human cells with T4 markers are cultivated in a substrate in the presence of the fluid sample, and then the cultivated cells are examined for cytopathic effect, c h a r a c t e r i z e d by cultivating the human cells with T4 markers as a monolayer culture in a substrate exchanged, if necessary, a plurality of times, the substrate in at least one of these exchanges containing the fluid sample. |
| 2. | A method according to claim 1, c h a r a c t e r ¬ i z e d by using human cells selected from a cell line of high affinity to LAV/HTLVIII virus. |
| 3. | A method according to claim 2, c h a r a c t e r ¬ i z e d by using human cells of the type lymphocytes, leukemia cells or cells from cerebral, lung, mucosa, hepatic or renal tissue. |
| 4. | A method according to any of claims 13, c h a r a c t e r i z e d by propagating the monolayer culture in a serumcontaining substrate for developing active cells, incubating the active cells with a serumfree substrate containing the fluid sample, and then culti¬ vating the cell culture with a substrate having a low content of serum. |
| 5. | A method according to claim 4, c h a r a c t e r ¬ i z e d by performing the cultivation of the monolayer structure having a low content of serum for a period of up to 30 days. |
| 6. | A method according to claims 15, c h a r a c t e r ¬ i z e d by examining the cells in the monolayer cul¬ ture microscopically for cytopathic effect after culti¬ vation. |
The present invention concerns a method of detecting infectious AIDS virus in human tissue fluids, such as blood or plasma.
The disease AIDS (acquired immunodeficiency syndrome) has only been known for a few years, but has developed almost explosively since 1981. At the end of 1984, more than 10,000 new cases were registered in the United States, but it is expected that another 10,000 cases will occur in 1985.
AIDS is caused by a virus called LAV (lymphadenopathy- associated virus) or HTLV-III (human T-cell lympho- tropic virus type III). This virus specifically in¬ fects T-4 lymphocytes, which play a central part in the control of the immunity system. The disease is particularly dangerous for immune deficiency patients because the patient is susceptiple to a large number of diseases to which he is normally immune. Examples are pulmonary diseases which develop mortally in such patients in a short time, and Kaposi's sarcoma which was previously an extremely rare disease, but which now attacks AIDS patients at a rate of more than 30_. It is also known that many AIDS patients suffer or have suffered from hepatitis.
Though the number, of patients with infectious AIDS increases rapidly, it has been noted that an even larger number of humans have developed antibodies against AIDS, without presenting any danger of infec¬ tion. It has also been found that humans may be in¬ fected with AIDS and thus present a danger of infec-
tion, without having yet developed antibodies against AIDS.
To try to limit the spreading of the disease, it is of great importance to detect all cases of infectious AIDS, so that infection from such patients can be pre¬ vented. Since infection particularly occurs by trans¬ mission or transfusion of blood or other tissue fluid from a carrier to another human, the possibility of detecting whether blood donors have infectious AIDS is of great importance.
So far, no method has been developed for detecting the presence of infectious AIDS, whereas it has been possible to detect antibodies against AIDS in tissue fluids.
A number of countries have taken measures to check all blood donors for the presence of antibodies against AIDS and rejected all positive ones. This, however, is no safeguard against the risk of drawing blood from AIDS-infected humans who have not (yet) developed antibodies. Further, it is not advisable to reject a large number of blood donors who, not withstanding that they have antibodies against AIDS, do not present any danger of infection.
The object of the present invention is to solve the above-mentioned problem by providing a method of de¬ tecting infectious AIDS in tissue fluid samples.
Another object of the invention it to detect whether infectious AIDS is present in injection preparations
It is known from Nature, vol. 316, 1985, p. 69-74 and 262-265, that virus produced from HTLV-III genome is infectious and exerts a significant cytopathic effect in vitro on T4 positive cells. Thus, human cells in suspension with T4 markers have been cultivated in a substrate containing HTLV-III virus. A cytopathic effect could be detected on the cells after cultiva¬ tion for some days.
However, the above-mentioned studies have not induced a skilled person to assume that it would be possible to develop a safe method of detecting infectious AIDS in tissue fluid samples by cultivating human cells with T4 markers in a substrate.
The present invention is based on the idea that a ono- layer culture of human cells with T4 markers may be used for specifically identifying infectious AIDS virus in tissue fluids and other fluid samples. It has sur¬ prisingly been found that this provides a very safe identification and a rational and simplified procedure. The method, which is of the type stated in the intro¬ ductory portion of claim 1, is thus characterized by the subject-matter in the characterizing portion of the claim.
The invention also concerns the use of human cells selected from a cell line of high affinity to LAV/HTLV- III virus. Such cells may be obtained by treating and cultivating lymphocytes or leukemia cells, but useful cells may also be obtained from cerebral, lung, mucosa, hepatic or renal tissue.
According to a preferred embodiment of the invention, the monolayer culture is propagated in a serum-contain-
ing substrate for developing active cells, and then the active cells are incubated with a serum-free substrate containing the fluid sample, followed by the cultiva¬ tion of the cell culture with a substrate having a low content of serum. Depending upon the amount of present LAV/HTLV-III virus, a cytopathic effect on the culti¬ vated cells may be detected after a certain period of time. The detection may take place microscopically, but any other detection method may also be used. Ex- amples of this are specific colour reactions or
1) fixation, colouring and microscopy
2) electron microscopy
3) determination of viral nucleic acid by a) specific colouring b) specific inhibitors and nucleic acid synthesis
4) plaque-assay method
5) flurorescent antibody test a) direct method b) indirect method 6) reverse transcriptase activity measurement.
The methods mentioned in items 1, 3, 4 and 5 are des¬ cribed by Robert J. Kuchler: "Biological Methods in Cell Culture and Virology", 1977, Dowdon, Hutchinsom & Ross, Juc.
It has been shown that certain virus types closely related to LAV/HTLV-III cause the same or a similar cytopathic effect on human cells with T4 markers. Examples of this are vira of the visna type, such as Maedi-visna virus which attacks sheep, see Nature New Biology, vol. 237, May 24, 1972, p. 114-115. LAV/HTLV- III virus is now classified as belonging to the sub¬ family lenti-virus, which also includes Maedi-visna
virus, the RNA structure and the morphological proper¬ ties being closely related.
A sample with a known content of AIDS virus may be used as a positive control in the present method of detecting AIDS virus. However, instead, a virus having correspond¬ ing properties may be used, such a Maedi-visna. This obviates the risk of infection of the laboratory staff with AIDS virus from the reagents and aids used.
The method of the invention will be illustrated more fully below by means of examples.
EXAMPLE 1
The selected cell line with T4 markers is inoculated into a multi-dish cultivation bowl with 24 holes, the same number of cells being inoculated into each hole so that 2/3 of confluent are obtained in 2 days. After a suitable growth (2/3 confluent), each hole is flushed 3 times with a growth medium containing peni¬ cillin and streptomycin, but without any content of seru .
The culture is incubated with the 3rd portion of growth medium which is added for 10 minutes at 37°C, and then the growth medium is removed. Following this, the holes are treated individually in the following manner:
1st row of holes (4 holes for positive control) are provided with 1 ml of growth medium without serum con¬ tent, but with 2-fold dilutions of LAV/HTLV-III virus.
2nd row of holes (4 holes for negative control). To each hole are added 0.5 ml of standardized sample mate-
rial found to be free of virus, and 0.5 ml of growth medium without serum.
3rd-6th rows (test rows). 0.5 ml of sample material and 0.5 ml of growth medium without serum are added to 4 . test holes in each row.
The cultivation bowls are incubated at 37°C for 4 hours. Then all liquid is sucked out of all holes. Then, 3 ml of antibiotics (penicillin and streptomycin) and 2_ fetal calf serum are added to ' each hole. The cultivation bowls are then incubated at 37°C for up to 30 days.
Media exchange with growth medium containing antibiotics and 2_ fetal calf serum is effected 3 times per week.
Each day cytopathic effect (CPE) is microscopically inspected and graded according to the following scale:
0 - confluent, normal healthy cells, no CPE 1+ - isolated "foci" and cells with CPE 2+ - about 50?ά of the cells has CPE 3+ - complete destruction of cell layers.
EXAMPLE 2
The procedure described in example 1 is repeated with the change that Maedi-visna virus is used instead of LAV/HTLV-III virus for positive control.
Examinations of a number of test samples from blood serum by the methods described in examples 1 and 2 gave the same result.
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