The novel human retrovirus named human T-cell lymphotropic virus type III ... Then, 1 ml of the virus-cell mixture was incubated for 2, 3,. 4, and 6 days.
JOURNAL OF CLINICAL MICROBIOLOGY, Dec. 1985, p. 908-911
Vol. 22, No. 6
0095-i137/85/120908-04$02.00/0 Copyright © 1985, American Society for Microbiology
Effect of Heat and Fresh Human Serum on the Infectivity of Human T-Cell Lymphotropic Virus Type III Evaluated with New Bioassay Systems SHINJI HARADA,* HIRONORI YOSHIYAMA,
AND
NAOKI YAMAMOTO
Department of Virology and Parasitology, Yamaguchi University School of Medicine, Ube, Yamaguchi, 755 Japan Received 19 June 1985/Accepted 20 August 1985
MT-4 cells, which are a human T-cell Iymphotropic virus type I (HTLV-I)-positive cell line highly permissive to HTLV-III infection, were used to detect the biologically active virus. For quantitation of the virus, induction of HTLV-III-specific antigen(s) and inhibition of DNA synthesis in infected MT-4 cells were assessed by indirect immunofluorescence and by a proliferation assay measuring [3H]thymidine uptake, respectively. HTLV-III was fully inactivated by treatment at 56°C for 30 min. It was not inactivated by treatment with fresh anti-HTLV-III-negative serum. Thus, these assay systems with MT-4 cells would be useful in further studies on
acquired immune deficiency syndrome.
The novel human retrovirus named human T-cell lymphotropic virus type III (HTLV-III), a lymphadenopathy-associated virus (LAV) frequently can be isolated from patients with acquired immune deficiency syndrome (AIDS) and AIDS-related complex (1, 10). Accumulated evidence suggests that the retrovirus is the cause of AIDS. Inactivation of the virus by chemical disinfectants (11) and heat (12) has been reported previously. However, it is still important to test quantitatively how the infectious virus can be inactivated, because reverse transcriptase (RT) activity, which was usually used as an indicator of the amount of virus, is not quantitatively accurate. It is indeed credible that the virus spreads through commercially available products from blood which has been contaminated with the AIDS virus (3, 4, 9). Recently, we found that the HTLV-I-carrying cell line, MT-4, was highly permissive to infection by HTLV-III; which is a prototype of the AIDS viruses (5). The virus-infécted MT-4 cells promptly produced virus-specific antigens and showed remarkable cytopathic effects. This relationship between the virus (HTLV-III) and the cells (MT-4) prompted us to develop a quantitative assay for the active virus and virus-infected cells. In fact, we have already established the HTLV-III and HTLV-III-infected cellinduced plaque forming assay with chemically adhered MT-4 cells (5, 6). Also, this system is available for the assay of the biological infectivity of the virus by a proliferation assay of infected MT-4 cells. In this study, we examined the inactivation of the virus by heat (56°C) and fresh human serum (complement), by using a newly established method for the quantitation of the virus.
anti-HTLV-III antibodies detected by immunofluorescence (IF) (10) in the serum. IF method. MT-4 cells were adjusted to a concentration of 106 per ml, and 0.5-ml samples of the cell suspensions were mixed with the same volume of treated and untreated virus. Twofold-diluted virus preparations were used as controls. Then, 1 ml of the virus-cell mixture was incubated for 2, 3, 4, and 6 days. Cell smears were prepared, dried, and fixed with cold methanol for 2 to 3 min. Fixed cells were then incubated with a 1:1,000 dilution of human anti-HTLV-IIIpositive serum (IF titer, 1:4,096) for 30 min at 37°C. They were washed three times with phosphate-buffered saline. The fluorescein-conjugated antiserum to human immunoglobulin G (Dakopatts A/S, Copenhagen, Denmark) was applied to the fixed cells for 30 min at 37°C, and they were washed again with phosphate-buffered saline. The IFpositive cells were examined under a fluorescence microscope. More than 500 cells were counted to calculate the percentage of positive cells. Proliferation assay. MT-4 cells were diluted to 6 x 105 cells per ml, and 50-,ul volumes of the cell preparations weré incubated with the same volume of treated and untreated virus at 37°C in a humidified CO2 incubator. After incubation for 3 days, cells were fed with 100 ,ul of fresh RPMI 1640 medium supplemented with 10% fetal bovine serum and antibiotics. On day 4 postinfection (p.i.), 1 ,uCi of tritiated thymidine (20.0 Ci/mM; New England Nuclear Corp., Boston, Mass.) was added to each well and incubated overnight. Cultures were harvested and counted in a scintillation counter by a standard technique. All experiments were performed in triplicate. Standard deviations of each experiment rarely exceeded 10% of the mean.
MATERIALS AND METHODS
RESULTS Heat inactivation of the virus. First, infectivity of the unheated control virus preparation was assessed for the induction of HTLV-III-specific antigen(s) detected by IF in MT-4 celis (Fig. 1B). On day 2 p.i., the number of IFpositive cells was determined and was found to have a linear correlation to the dilution of the virus. Thus, the number of IF-positive cells is an expression of the amount of biologically active virus. On the basis of these data, the virus was used without dilution for further experiments. HTLV-III
Virus. HTLV-III was obtained from the filtered supernatant fluid of H9-HTLV-III cells. The titer of HTLV-III used here was 4 x 103 PFU/ml. The virus was heated at 56°C in a water bath for 1, 2.5, 5, 10, 15, 30, and 60 min. Heating was stopped by putting the samples in an ice bath. The virus was also incubated with fresh and decomplemented (56°C, 30 min) human serum for 30 min at 37°C. There were no *
Corresponding author. 908
INACTIVATION OF HTLV-III
VOL. 22, 1985
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10 15 30 60 01255 MINUTES EXPOSED M HEAT
/2 1
VIRUS
FIG. 1. (A) Effect of different exposure times of HTLV-III to heat on the induction of HTLV-III-specific antigen(s) in infected MT-4 cells. (B) Kinetic induction of HTLV-III-specific antigen(s) in infected cells when serial twofold-diluted preparations of the virus were used as the standard concentrations. IF was performed on days 2 (G), 3 (0), 4 (O), and 6 (X) p.i. No fluorescent cells were observed in uninfected MT-4 cells at any stage of culture.
was treated with heat at 56°C for 1, 2.5, 5, 15, 30, and 60 min. The effects of the treatment on the induction of HTLV-IIIspecific antigens is shown in Fig. 1A. The percentage of IF-positive cells in MT-4 cells infected with nontreated virus
909
(O min) was 12.2% 2 days p.i., whereas the percentage of those infected with the virus heated for 2.5 min was 1.5%. Reduction of viral infectivity after heat treatment for 2.5 min was 88%. Infected MT-4 cells were monitored daily for the appearance of IF-positive cells until 6 days p.i. The sensitivity of detection of the virus became higher, because infected MT-4 cells released virus progeny which infected neighboring uninfected MT-4 cells in the cultures. In fact, on days 4 and 6 p.i., nearly 100% of the cells were fluorescent even in 1/16-diluted virus-infected cells. On day 6 p.i., no IF-positive cells were observed when the virus was treated for 30 min or more. Thus, heating the virus at 56°C for 30 min appeared to be sufficient to inactivate its infectivity (Fig. 1A). Next, to further confirm virus inactivation by heating, a proliferation assay was used, because this assay was more quantitative and objective than IF. The HTLV-III-induced cytopathic effect was inversely expressed by the uptake of [3H]thymidine. The viral dose response was linear when the proliferation assay was performed 5 days p.i. (Fig. 2). Reduction of viral activity by heating was calculated to be 50 and 90% at 1 and 2.5 min, respectively. Thus, the virus was rapidly inactivated by heating at 56°C. A proliferation assay was also performed 7 days p.i. to detect the effect of a minute amount of living virus. Inhibition of DNA synthesis detected by the proliferation assay was also observed in the MT-4 cells infected with virus treated for 10 min (data not shown). If both assays (IF and proliferation) were performed on the same day p.i., the sensitivity of the proliferation assay was lower than that of IF, because appearance of the cytopathic effect was always preceded by the production of virus-specific antigen(s) (S. Harada, Y. Koyanagi, and N. Yamamoto, Virology, in press). Effect of fresh serum negative to HTLV-III on the infectivity of the virus. Fresh serum (mainly complement) did not affect the infectivity of the virus (Table 1). IF and proliferation assays were performed 2.5 and 5 days p.i., respectively. The IF positivity and radioactivity of infected MT-4 cells were
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011 Z5 5
30 60 MINUTES EXPOSED TO HEAT 10
15
1
DILUTION OF VIRUS
FIG. 2. (A) Effects of different exposure times of HTLV-TII to heat on the DNA synthesis of the infected MT-4 cells. Panel B shows that the viral dose response was linear as detected by the DNA synthesis of infected MT-4 cells. The proliferation assay was performed on day 5 after infection.
910
HARADA ET AL.
J. CLIN. MICROBIOL.
TABLE 1. Effects of human serum on the infectivity of HTLV-III Treatment
Virus control
Medium only Heated serum Fresh serum
Anti-HTLV-IIIpositive
Dilution
Dilution
1 1/4
[HJthymidine
IF-positive
tells () r-ells
M
1/10
22.0 6.4 1.4
1/5 1/10 1/20 1/5 1/10 1/20 1/20
0 20.4 25.7 34.7 22.6 28.7 29.7 1.0
~
uptakea
(X 10-4)
1.8 3.3 21.7 24.9
± 0.1 ± 1.2 ± 0.3 ± 0.2
1.2 1.4 1.7 1.2 1.1 1.3
± 0.07 ± 0.03 ± 0.3 ± 0.2 0.3 ± 0.08
NTb
serum
a proliferation assay and virus-specific antigen induction of infected MT-4 cells. The effect of other means of physical and chemical inactivation of the AIDS retrovirus, such as UV and chemical disinfectants, could be studied in a similar manner. In all cases, these studies should be conducted taking into account the degree of inactivation of constituents such as factor VIII in the blood products. Such studies are under way in our laboratory. Using the methods described above, we studied the effect(s) on the infectivity of HTLV-III of fresh human serum from an individual seronegative for HTLV-III. Unlike with other animal retrovirus (13), viral infectivity was not affected by fresh human serum. These results indicate that HTLV-III resembles HTLV-I in its resistance to human complement (7). This may be a common feature of human retroviruses which share tropism for OKT4-positive lymphocytes (8, 14). The finding that HTLV-III is not lysed by human complement may explain why the virus can be transmitted through contaminated blood or blood products.
(heated) a [3H]thymidine uptake is expressed as mean counts per minute ± standard deviation. b NT, Not tested.
again linearly dependent on the amount of virus. Fresh antibody-negative serum was not seen by either method to inactivate the virus. However, anti-HTLV-III-positive serum used as the positive control neutralized 90% of the virus even at a 1/20 dilution. DISCUSSION Increasing evidence, mainly obtained from seroepidemiological studies, has suggested that blood products such as factor VIII or IX are the cause of the high prevalence of antibodies to the AIDS retrovirus in hemophiliacs who receive them (3, 4, 9). Thus, it is urgent to develop a simple, rapid, and sensitive screening test for determining the presence or absence of the virus in such blood products. Spire et al. used the measurement of RT activity to study the effect of heating on the LAV (12). They also examined LAV infectivity in human cord blood lymphocytes by measuring viral production as determined by RT activity (12). There are several problems with their assay systems. For example, RT activity does not represent the amount of virus, especially in terms of viral activity. In view of that, even a single infectious virion can multiply to several magnitudes, increasing the amount of virus in the infected cells, if the appropriate circumstances are provided. It is most important to be able to rhe ssUre the residual activity of even small amounts of infectious viruses after physical or chemical treatment of blood products. Furthermore, phytohemagglutinin-stimulated primary lymphocytes, which were used as target cells for LAV infection, are highly heterogeneous, so that thé test for LAV infectivity is neither reproducible nor sensitive. In this context, our procedure can indeed measure the biologically active virus by using the highly homogeneous cell line MT-4. Moreover, our systems, especially the proliferation assay of virus-infected MT-4 cells, are advantageous from the standpoints of rapidity, simplicity, and sensitivity, as well as reproducibility for quantitative analysis of the virus. Spire et al. previously reported that the RT activity of LAV was completely abolished by heating at 560C for 30 min in their system (12). The same results were obtained with infectivity assays (2). Our results confirmed these findings by
ACKNOWLEDGMENTS We thank Y. Koyanagi for typing the manuscript and the staff in our laboratory for encouragement and advice. This work was supported by a Grant-in-Aid for Cancer Research from the Ministry of Education, Science and Culture.
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