Acid Stability of Hepatitis A Virus

Transcription

1 J. gen. Virol. (1989), 70, Printed in Great Britain 2481 Key words: acid stability~hepatitis A virus/enteroviruses Acid Stability of Hepatitis A Virus By ELKE SCHOLZ, URSULA HEINRICY AND BERTRAM FLEHMIG* Abteilung fur Medizinische Virologie und Epidemiologie der Viruskrankheiten, Hygiene-Institut der Universitiit Tiibingen 74 Tiibingen, Silcherstrasse 7, F.R.G. (Accepted 5 May 1989) SUMMARY The acid stability of unpurified and highly purified hepatitis A virus (HAV) was tested and compared with that of poliovirus type l, coxsackievirus types A9 and B 1 and echovirus type 9. Only HAV had a high residual infectivity after 2 h of exposure to ph 1 at room temperature, remaining infectious for up to 5 h. At 38 C, ph 1, HAV remained infectious for 90 min. Highly purified HAV was found to be infectious for 8 h at ph 1 and room temperature. This indicates that the increased stability is not due to protection by cellular material attached to the virus, but is a virus-specific marker. Under the same conditions, at ph 1 and room temperature, unpurified and highly purified HAV antigens were traceable for 5 and 4 h respectively. INTRODUCTION It is known that all enteroviruses including hepatitis A virus (HAV) show stable infectivity at ph 3 (Newman et al., 1973; Siegl et al., 1984). The effect of lower ph values has been examined for only a few enteroviruses. Poliovirus type 1 (PV l) and coxsackie B viruses were shown to be relatively unstable at ph 1. Loring & Schwerdt (1944) found that the infectivity of PV 1 was completely destroyed within 15 to 20 min when exposed to ph 1 at room temperature, and a more detailed study showed that the Lansing strain of poliovirus was totally inactivated after 5 min at 37 C and ph 1 (Faber & Dong, 1946). Coxsackie B viruses lost all their infectivity after l day of exposure to ph 1 at room temperature (Robinson, 1950). For HAV, it has been recently stated that there was a loss of stability at ph values less than 2 (Coulepis et al, 1987). In this study we have compared the stability of PV l, coxsackievirus type A9 (CV A9), coxsackievirus type Bl (CV B1), echovirus type 9 (EV 9) and HAV towards ph 1 at room temperature in more detail and found that HAV exhibited an extraordinary stability at low ph. Since HAV is known to attach large amounts of cellular material to its surface (Lemon & Binn, 1985; Heinricy et al., 1987), possibly protecting itself from degradation, we have also examined highly purified HAV at ph l and were able to show that it was even more stable. We also studied the effect of ph 1 on the purified and unpurified HAV antigen. The stability of infectious HAV at ph l was also tested at 38 C, which represents the temperature of the interior of the human body. The result is discussed in the light of the knowledge that ph 1 represents the acidity of the digestive secretions (Pschyrembel, 1969; Faber & Dong, 1946), to which all enteroviruses are exposed in the course of a natural infection. METHODS Preparation of viruses. CV A9, CV B1 and EV 9 were propagated in HEp-2 cells and PV 1 (vaccine strain) was grown in BGM cells. After the removal of cell debris, the viruses were concentrated by centrifugation at g for 4 h. The pellets were resuspended in phosphate-buffered saline (PBS). For HAV, the supernatant of human embryonic lung fibroblasts persistently infected (Vallbracht et al., 1984) with HAV strain HAV/HFS/GBM (Flehmig et al., 1981) was ultracentrifuged for 15 h at g and the virus pellet was resuspended in PBS SGM

2 2482 E. SCHOLZ, U. HEINRICY AND B. FLEHMIG Purification of HA V. Human embryonic lung fibroblasts infected with HAV strain HAV/HFS/GBM, passaged 35 times in human fibroblasts, were dissolved with versene-trypsin. After removal of cell debris by centrifugation for 20 min at 2000 g, HAV was concentrated by tangential flow filtration using the Minitan System (Millipore). HAV adhering to the cell debris was recovered by multiple sonications and, after removal of cell fragments by centrifugation (20 min, 2000 g) and sterile filtration, added before the concentration step. After ultracentrifugation for 15 h at g at 4 C, the HAV pellet was resuspended in PBS by brief sonication (1 to 3 min). HAV was freed from lipid by preincubation at ph 3 (achieved by the addition of 1 M-HCI) at a temperature of 37 C for 2 h and subsequently centrifuged through a discontinuous sucrose gradient [30~ and 60~ sucrose in TNE buffer (10 mm-tris-hc1, 150 mm-n acl, 1 mm-edta, ph 7.4; Provost et al., 1986) plus 0-5 ~ sodium lauryl sarcosinate]. The gradient was fractionated and the protein content of the fractions was determined by the method of Lowry et al. (1951). The HAV antigen content of the fractions was determined by radioimmunoassay (RIA; Flehmig et al., 1978). HAV-positive fractions were pooled and dialysed against PBS. To demonstrate the purity of the HAV preparation, SDS-PAGE (Laemmli, 1970) with subsequent silver staining (Ansorge, 1983) was carried out. The gel electrophoretic analysis showed three of the viral protein bands and only minimal impurities (data not shown). Acid treatment and neutralization. The acid treatment was performed at room temperature, 24 C _+ 3 C, with gentle stirring of the solution or at 38 C C without stirring. Adjustment to ph 1 was achieved by diluting the viral material 100-fold in KC1-HC1 buffer giving a final concentration of 0.15 M. Controls were diluted 100- fold in doable distilled water or PBS. The ph was determined using a glass electrode and found to be in the range 1"02 to 1.04 at room temperature or 0.96 at 38 C. After different periods of incubation aliquots were taken, diluted 10-fold in maintenance medium plus the required amount of foetal calf serum, neutralized by the addition of 1 M- NaOH, monitored by ph indicator paper (Merck) and diluted further in maintenance medium supplemented by foetal calf serum. Determination of residual infectivity. Residual infectivity was titrated by inoculating eight wells of a 96-well microtitre plate containing a dense monolayer of susceptible cells with 200 p.l of each 10-fold dilution of virus. PV 1 was titrated on BGM cells, EV 9, CV A9 and CV B1 on HEp-2 cells and HAV on human embryonic fibroblasts. The plates were incubated at 37 C, in 5~ CO2, and screened for the presence of c.p.e, after 6 days for PV 1, CV A9 and CV B1 and after 10 days for EV 9. For HAV the cells were frozen and thawed three times after an incubation period of 21 days and the supernatants were tested in a RIA for the HAV antigen as described (Flehmig et al., 1978). The TCIDso/ml was calculated by the method of Karber (1931). Determination of residual antigenicity of purified and unpurified HA V. HAV was diluted fourfold in a KC1-HC1 buffer to a final concentration of 0.15 M. Measurement with a glass electrode showed a ph value of 1"04. During incubation at room temperature the solution was gently stirred. Aliquots taken after different incubation periods were directly neutralized by the addition of 1 M-NaOH, monitored by ph paper, and further diluted in PBS for the determination of residual antigenicity by RIA. RESULTS Residual infectivities (TCIDs0/ml) of the five viruses tested for their stability at ph 1 at room temperature are presented in Table 1. As shown, HAV exhibited an outstanding stability at ph 1, remaining infectious for up to 5 h. CV A9 and CV B1 were slightly more stable than PV 1 and EV 9 which were totally inactivated in less than 10 and 30 rain, respectively. Further experiments with highly purified HAV showed that after purification the stability of HAV at ph 1 was increased. In Fig. 1 the decline of infectivity of highly purified and unpurified HAV preparations at ph 1 and room temperature are compared. Highly purified HAV remained infectious for up to 8 h at ph 1. This finding indicates that cellular material attached to the HAV surface does not account for the high acid stability of HAV. In order to obtain more insight concerning the mechanism of inactivation at ph 1 the stability of the HAV antigen was studied. Highly purified and unpurified HAV antigen was traceable for 4 and 5 h, respectively (Table 2), showing high resistance towards degradation. In both preparations breakdown of the antigen structure was initiated more rapidly than the breakdown of infectivity. At 38 C, which represents the temperature of the interior of the human body, unpurified HAV remained infectious at ph 1 for up to 90 min (Fig. 2). Within the first 60 min only slow degradation occurred, leaving 10~ of the initial amount of infectious HAV intact.

3 Acid stability of HA V 2483 l0 7! i i! i i i! ~ 10 4 ~ 10 3 'N.~ 10 4 > ".,~..= ~ 10 3.~ 10 2, F" ~ 10 2 lo'1- I 'k -t 10' 10 " ~ 10 A Time (h) Fig Time (h) Fig. 2 Fig. 1. Decline of highly purified (A) and unpurified (V) HAV at ph 1, at room temperature. Highly purified HAV exhibited higher stability than unpurified HAV, showing that the cellular material attached to the virus did not account for the extraordinary acid stability of HAV in comparison to other enteroviruses. Fig. 2. Stability of unpurified HAV at ph 1 and 38 C. Table 1. Residual infectivity of the five enteroviruses tested for stability at ph 1 and room temperature Titre of infectivity [loglo(tcidso/ml)] A Time HAV CV A9 CV B1 PV 1 EV 9 0 min min 6.7 -* min min min min h h h h h h h h 4.t h h h h h h * -, Not determined.

4 2484 Table 2. E. SCHOLZ, U. HEINRICY AND B. FLEHMIG Titre of residual antigenicity of unpurified and highly purified HA V after incubation at ph 1 at room temperature for different lengths of time Titre of residual antigenicity* A Time Unpurified HAV Highly purified HAV 0 min 1:64 1:32 10 rain -t 1 : rain 1:32 1 : h 1:32 1:16 l-5 h 1:16-2-0h 1:16 1:8 2.5h 1:16-3.2h 1:16-3.7h 1:8-4.0h 1:8 1:4 5.0 h 1:4-6.0h <1:4 8.0h <1: h < 1:4 * Titres are expressed as the highest dilution giving a positive result in the RIA. t -, Not determined. DISCUSSION Our results show that in comparison to other enteroviruses, HAV exhibits an extremely high stability at low ph. Concentrated cell culture supernatant HAV was still infectious after 5 h of exposure to ph 1 at room temperature, whereas other enteroviruses had lost nearly all of their infectivity within 2 h. The possibility that the increased stability of HAV at low ph was caused by a protective effect from large amounts of cellular material attached to the HAV surface was excluded by testing highly purified HAV, which showed an even greater stability at low ph and remained infectious for up to 8 h at ph 1 and room temperature. Adjustment to ph 1 was achieved with a 0.15 M-KCI-HCI buffer solution. Examinations by Salo & Cliver (1976) have shown that N ac1 and other chloride salts enhance the inactivation of poliovirus type 1 at around ph 3 at concentrations as low as 0.1 M. Although it has been described that 0.1 to 0.2 M-KC1 had no enhancing effect on the inactivation of HAV at ph 4 to 7 (Siegl et al, 1984), it is possible that not only H + but C1- also plays a role in the inactivation of the viruses at lower ph values. Little is known about the mechanism of inactivation of viruses at ph 1. Salo & Cliver (1976) discussed the possible mechanisms of destruction of the viral protein coat and RNA. By examining the effect of ph 1 on purified and unpurified HAV we found that initially the degradation of antigenicity occurred faster than the degradation of infectivity. Later in the course of degradation the kinetics altered and loss of infectivity occurred faster than the loss of antigenicity. The parallel course of antigen breakdown in both preparations indicates that cellular material has its main effect on viral infectivity, reducing the stability of unpurified infectious HAV. Faber & Dong (1946) discussed their results of inactivation of poliovirus at phl at 37 C in connection with gastric digestion of the virus. They concluded that during fasting and at the height of digestion of carbohydrates and mixed meals containing meat, poliovirus was rapidly inactivated because of the high levels of acidity. Proteolytic degradation seemed to play a minor role. Since the pathway of pathogenesis for HAV is still uncertain, our finding of the extraordinarily high acid stability of HAV, even at 38 C, might be of some importance. Taking into account that evacuation of the stomach begins within 1 to 7 min after digestion (depending on the kind of food taken) our finding could imply that HAV does not necessarily need a multiplication step in the oropharynx before passage through the acid environment of the 'digestive system as other enteroviruses do (Lonberg-Holm & Philipson, 1981 ; White & Fenner, [986) in order to ensure that enough virus reaches the intestinal tract to initiate an infection. If

5 Acid stability of HA V 2485 this assumption is correct the primary uptake of HAV could be intestinal, and not pharyngeal as described for other enteroviruses. In fact, mutiplication of HAV in the throat of humans has not been demonstrable. With regard to the development of an attenuated, live hepatitis A vaccine our results point to the great importance of testing the acid stability of the vaccine strain when oral application is considered. By this route low amounts of attenuated virus might be sufficient to achieve immunity in humans, giving rise to high IgA titres in the gut. The results of this study are further evidence that HAV is an enterovirus with unusual behaviour, and therefore may need to be reclassified within the Picornavirus group. REFERENCES ANSORGE, W. (1983). Fast visualization of protein bands by impregnation in potassium permanganate and silver nitrate. In Electrophoresis '82, pp Edited by D. Stathakos. Berlin & New York: Walter de Gruyter. COULEI, IS, A. G., ANDERSON, 13. N. & GUST, I. D. (1987). Hepatitis A. Advances in Virus Research 32, FABER, n. K. & DONG, L. (1946). Inactivation of poliomyelitis virus in relation to gastric and intestinal digestion. Proceedings of the Society for Experimental Biology and Medicine 63, rlemait3, B., RANKE, M., FRAr~X:, H. & GERTrt, n. J. (1978). Application of a solid-phase radioimmunoassay and immune electron microscopy for hepatitis A in diagnosis and research. Medical Microbiology and Immunology 166, FLEr~IIG, a., VALLBRACrrr, A. & WURSTER, G. (1981). Hepatitis A virus in cell culture. III. Propagation of hepatitis A virus in human embryo kidney ceils and human embryo fibroblast strains. Medical Microbiology and Immunology 170, HEINRICY, U., STIERHOF, Y.-D., PFISTERER, M. & FLEHMIG, B. (1987). Properties of a hepatitis A virus candidate vaccine strain. Journal of General Virology 68, K.~BER, G. (1931). Beitrag zur kollektiven Behandlung pharmakologischer Reihenversuche. Archiv J~r experimentelle Pathologic und Pharmakologie 162, LAEMMLI, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, London 227, LEMON, S. M. & BINN, L. N. (1985). Incomplete neutralization of hepatitis A virus in vitro due to lipid-associated virions. Journal of General Virology 66, LONBERG-HOLM, K. & PHILIPSON, L. (editors) (1981). Pathogenesis of picornaviruses. In Virus Receptors, vol. 2, pp London & New York: Chapman and Hall. LORING, n. S. & SCHWERDT, C. P. (1944). Studies on purification of poliomyelitis virus. II. ph stability range of MVA strain. Proceedings of the Society for Experimental Biology and Medicine 57, LOWRY, O. H., ROSEBROUGH, N. J., FARR, A. L. & RANDALL, R. J. (1951). Protein measurement with the Folin phenol reagent. Journal of Biological Chemistry 193, NEWMAN, J. F. E., ROWLANDS, D. J. & BROWN, F. (1973). A physico-chemical sub-grouping of the mammalian picornaviruses. Journal of General Virology 18, PROVOST, P. J., HUGHES, J. V., MILLER, W. J., GIESA, P. A., BANKER, F. S. & EMINI, E. A. (1986). An inactivated hepatitis A viral vaccine of cell culture origin. Journal of Medical Virology 19, I'SCnYREMBEL, W. (1969). Klinisches Wi~rterbuch, p Berlin & New York: Walter de Gruyter. ROBINSON, L. K. (1950). Effect of heat and of ph on strains of Coxsackie virus. Proceedings of the Society for Experimental Biology and Medicine 75, SALO, R. J. & OLIVER, D. O. (1976). Effect of acid ph, salts, and temperature on the infectivity and physical integrity of enteroviruses. Archives of Virology 52, SlEGL, G., WEITZ, M. & KRONAUER, G. (1984). Stability of hepatitis A virus. Intervirology 22, VALLBRACHT, A., HOFMANN, L., WURSTER, K. G. & FLEHMIG, B. (1984). Persistent infection of human fibroblasts by hepatitis A virus. Journal of General Virology 65, WHITE, D. O. & FENNER, F. J. (editors) (1986). Pathogenesis and pathology of viral infections (chapter 5), Picornaviruses and calciviruses (chapter 19). In Medical Virology, pp , Orlando: Academic Press. (Received 2 February 1989)