Journal of Medical Virology 37:174-179 (1992)

Accumulation and Persistence of Hepatitis A Virus in Mussels

Ricardo Enriquez, Gert Gustav Frosner, Volker Hochstein-Mintzel, Stella Riedemann, and German Reinhardt Instituto de Microbiologia, Universidad Austral de Chile, Valdiuia, Chile (R.E., V,H.-M., S.R., G.R.), and Max von Pettenkofer-lnstitut, University of Munich, Munich, Germany (G.G.F.)

Accumulation and persistence of hepatitis A vi- rus (HAV) in the mussel Mytilus chilensis was evaluated. Under optimal filtration activity of mussels (temperature 12”C, salinity 3%, feeding twice a day with Dunaliella marina), HAV was concentrated 100-fold from the surrounding wa- ter. Similar concentrations of HAV were reached in the filtration apparatus and in the digestive system (hepatopancreas). HAV persisted for about 7 days in mussels. Elimination of HAV from mussels was slower than elimination of po- liovirus. Without feeding of mussels (causing low filtration activity), there was no measurable uptake of HAV into mussels, and depuration of HAV from mussels was slower. The ability of mussels to concentrate HAV was used success- fully to monitor fecally contaminated river water for the presence of HAV. Q 1992 Wiley-Liss, Inc.

KEY WORDS: hepatitis A virus, shellfish, mus- sel, concentration, persistence, depuration

INTRODUCTION Most epidemics associated with shellfish are related

to hepatitis A or viral gastroenteritis [Levin, 1978; Gerba and Goyall, 19781. In many countries, ingestion of contaminated shellfish is an important mode of transmission of hepatitis A infection. In Shanghai, be- tween January and April 1988 a total of 292,301 per- sons suffered from clinical hepatitis A, and most of these had eaten clams IHalliday et al., 19911. In Ger- many, about one-third of patients with clinical hepati- tis A provide a history of recent shellfish consumption [Stille et al., 19721. However, most data available on concentration and persistence of viruses in shellfish have been obtained during studies of enteroviruses other than hepatitis A virus (HAV) [for review see Feachem et al., 19811. It is not known whether these data are also valid for HAV.

In 1979 techniques for the isolation of hepatitis A virus in tissue culture were introduced [Provost and Hilleman, 1979; Frosner et al., 19791. Using these tech- (G 1992 WILEY-LISS. INC.

niques, accumulation and persistence of HAV in shell- fish were studied after concentration of virus in Mytilus chilensis from surrounding water. Tissue culture grown HAV as well as HAV present in contaminated river water was monitored. Persistence of HAV and poliovi- rus is compared.

MATERIALS AND METHODS Origin of Mussels

The mussels used in this study (Mytilus chilensis) were harvested from natural banks of the Valdivia river estuary (altitude 39”48‘ south, latitude 73’14’ west) in Chile. Depuration was achieved by keeping the mussels in culture for at least 10 days at 12°C. Filtered sea water (Millipore filter with pore size 0.3 p,m were used) with a salinity of 3% was changed daily, and mussels were fed twice a day with 2 x lo7 cells of Du- naliella marina. The photoperiod was 12 hours. Only mussels with a shell length of 55-60 mm were used. Under these optimal conditions, each mussel filters be- tween 1.32 and 1.52 liters of water an hour lNavarro and Winter, 19821.

Origin of Hepatitis A Virus Hepatitis A virus (HAW from two sources was used.

In most experiments, tissue-culture grown HAV pro- duced in PLCiPRF 5 cells was used for feeding of mus- sels [Frosner e t al., 19791. After infection with the 8th to 10th tissue culture passage of the German HAV iso- late MBB, tissue culture cells were propagated for 3 weeks. A virus suspension containing about lo6 tissue culture infectious doses 50 (TCID,,) per ml was ob- tained by three times freezing and thawing of infected cells present in a tissue culture box having 10 cell lay- ers on top of each other (“Wannenstapel,” Nunc, Heidel- berg, Germany) in the presence of 50 ml of minimum essential medium (MEM). The virus suspension was clarified by low-speed centrifugation (2,000 rpm for 10 minutes) before use.

Accepted for publication January 7,1992. Address reprint requests to Prof: G. Frosner, Max von Petten-

kofer-Institut, Pettenkoferstr. 9a, 8000 Munich 2, Germany.

Concentration of HAV in Mussels 175

1 161 @ refrigeration system @cirtulotion pump @limo clock

@ tnrrmostot @culture unit @ p h n t D period

@ ihsrmoregulotsd both @ a e r a t i o n pump @ eiectric outlet

Fig. 2. Experimental design for cultivation of Mytilus chilensis dur- ing studies investigating concentration and persistence of hepatitis A virus or poliovirus in the mussels.

Areas served by sewage outlets --- - - city limits ......... 0 Hospitals treating hepatitis A patients 0 Hospitals not treating hepatitis A patients * Universidad Austral de Chile

Fig. 1. Areas of the city of Valdivia served by the four sewage outlets investigated in this study. The two hospitals treating hepatitis A patients are connected t o the outlet of area I. In area 111 there are two other hospitals that do not treat hepatitis patients. River water inves- tigated for the presence of HAV was collected daily for 2 weeks 1 m from each of the four sewage outlets.

In one experiment, fecally contaminated river water collected near 4 different sewage outlets of the city of Valdivia was investigated. Using a tubing connected to a pump, for 2 weeks 1 liter of water was collected daily 1 m from each of 4 sewage outlets. The depth of collection varied with tidal changes. In Figure 1 the town areas of Valdivia served by the sewage outlets are shown. Two hospitals situated in the area served by outlet I treat hepatitis A patients regularly. Water samples were in- vestigated for the presence of HAV as described below.

Cultivation System for Mytilus chilensis During the experiments, 4-6 mussels were kept in

round aquariums 16 cm in diameter and 17 cm in height. Aquariums had been sterilized by UV-irradia- tion before use (Siemens UVL-q, 200 ergimm'). Each culture unit was filled with 1.0 liter of filtrated sea water with a salinity of 3%. Water was changed every day. Aeration was achieved by continuously pumping

air through a plastic tubing into the water of the aquar- iums. A constant temperature was maintained by locat- ing the culture unit in a tank containing 170 liters of water. The water temperature of this tank was cooled to 12"C, using a cooling system with a thermostat. In all experiments a photoperiod of 12 hours was chosen. Twice daily, 2 x lo7 cells of Dunaliella marina were added to the cultivation system for feeding of mussels. A model of the cultivation system is shown in Figure 2.

Titration of HAV Infectivity The infectivity of HAV was titrated in PLCiPRF 5

cells. Four tissue culture flasks were inoculated with 10-fold dilutions of mussel homogenates or of water aliquots (2 ml). When using tissue culture adapted HAV, tissue culture homogenates were tested for the presence of hepatitis A antigen by radioimmunoassay after 4 weeks of incubation [Frosner et al., 19791. For experiments with inoculation of fecally contaminated water the incubation time was 6 weeks. Cultures exhib- iting more than the 2.1-fold of the cpm found in nonin- fected cultures were considered positive. The number of TCID,, present in a sample was calculated according to Reed and Muench [ 19381.

A homogenate of the mussel was prepared by opening the mussel with a scalpel and scraping out the total contents of the shell. This material was then rinsed at room temperature three times for 10 minutes each in phosphate-buffered saline pH 7.2 (containing 100 IUirnl penicillin and 100 pgiml streptomycin). The main muscles (musculus adductor and the foot muscle) were eliminated and the remaining material (about 1 ml) was minced with a scalpel together with approxi- mately the same volume of Hanks' balanced salt solu- tion containing the above mentioned antibiotics. In some experiments, organs of the mussels were prepared and homogenized separately.

176 Enriquez et al.

Further homogenization was obtained by shaking the mixtures for 10 minutes with a Vortex-Genie mixer (Scientific Industries Inc., Bohemia, USA) a t speed number 7. Homogenized mussel samples as well as wa- ter samples were stored in liquid nitrogen until titra- tion of HAV.

RESULTS Concentration of HAV in Mytilus chilensis 24

Hours After Addition of Virus to the Surrounding Water

In this experiment, the culture system contained four mussels. To 1 liter of filtered sea water, 15 ml of HAV suspension with a n infectivity titer of lo6 TCID,, per ml (in total 1.5 x lo7 TCID,,) was added 5 minutes after feeding the mussels with Dunaliella marina. Af- ter 24 hours, the infectivity titer of HAV in the sea water was 3.2 x l o 3 per ml(3.2 x lo6 TCID,, in 1 liter of sea water present in the culture system). In the pooled mussel extracts 3.2 x lo5 TCID,, was detected per ml (2.6 x lo6 TCID,, in 8 ml of extract). In the pooled extracts of two noninoculated mussels kept un- der the same conditions no HAV could be found (less than 3.2 x lo1 TCID,, per ml of extract). Therefore, 24 hours after addition of HAV, the concentration of virus in shellfish was about 100-fold that of the surrounding water.

Time of Maximal HAV Concentration After Feeding of Virus to Mytilus chilensis

The speed of accumulation of HAV was tested by measuring the infectivity of mussel extracts at 3 and 24 hours after addition of HAV. To each culture system, 8 ml of tissue-culture grown HAV with an infectivity titer of lo6 TCID,, per ml was added (8 x lo6 TCID,, in total). The pooled extracts of two mussels kept in sepa- rate culture systems for the indicated times were ti- trated for infectious HAV in PLCiPRF 5 cell cultures.

After 3 hours, 2.1 x lo6 TCID,, was measured per ml of extract. The total amount of infectious units in the 4 ml of extract was calculated to be 8.4 x lo6. After 24 hours the infectivity found in the pooled extracts was 4.6 x 10, TCID,, per ml giving a total of 1.8 x lo6 in- fectious units present within the mussels. Obviously, under optimal filtration conditions maximal concentra- tion of HAV is reached in shellfish in less than 24 hours and probably even within 3 hours after the addition of HAV to the surrounding water.

Concentration of HAV in Hepatopancreas and Filtration Apparatus of Mytilus chilensis

Eight milliliters of HAV suspension (lo6 TCID,, per ml) was added to a n aquarium containing 6 mussels, under optimal filtration conditions. After 3 hours, the hepatopancreas which represents the digestive system of the mussel was separated from the rest of the mussel which mainly constitutes the filtration apparatus. HAV concentration was determined in homogenates of both. Hepatopancreas showed only a slightly lower

HAV concentration than the rest of the mussel (1.7 x lo4 and 4 x lo4 TCID,, per ml, respectively). After uptake of' HAV by the filtration apparatus HAV seems to move rapidly to the hepatopancreas.

Concentration of HAV in Mytilus chilensis Under Different Feeding Conditions

Five milliliters of tissue culture grown HAV (lo6 TCID,, per ml) was added to two aquariums containing 4 mussels each. In one aquarium, mussels were kept under optimal filtration conditions while mussels in the other aquarium were not fed, After 3 hours a ho- mogenate of the hepatopancreas from mussels fed with Dunaliella marina contained 5.9 x lo3 TCID,, per ml of HAV whereas HAV was not detected in the hepato- pancreas of mussels which had not been fed (less than 3.2 x 102TCID,, per ml).

After 7 days, HAV was still detectable in high con- centration in mussels fed twice a day with Dunaliella marina with daily change of water (3.2 x lo5 TCID,, per ml in hepatopancreas and 3.2 x lo3 TCID,, in ho- mogenate of the rest of the mussel). HAV was detect- able neither in hepatopancreas nor in the rest of the mussels not having been fed (less than 3.2 x lo2 TCID,, per ml. respectively). Obviously, without feed- ing there is no measurable increase in HAV concentra- tion of hepatopancreas and of filtration apparatus. Therefore, in experimental systems used for monitor- ing HAV concentration in shellfish different feeding conditions can be used to modify filtration activity and accumulation of HAV in mussels.

Persistence of HAV in Mytilus chilensis Under Different Feeding Conditions

Under optimal filtration conditions, 10 ml of tissue culture grown HAV (lo6 TCID,, per ml) was added to each of two aquariums containing 4 mussels. After 1 day, the HAV concentration in the extract of one mus- sel was 1.7 x 10, per ml. The water was then changed daily for 11 days. In one aquarium the mussels were fed twice a day with Dunaliella marina, while in the other aquarium mussels were not fed after the daily change of water.

In the group fed, HAV was still detectable in mussel extract after 7 days (1 x lo4 TCID,, per ml), but unde- tectable after 11 days (less than 3.2 x 10' TCID,,). Without feeding, the clearance of HAV from mussels was much slower (4.8 x lo5 TCID,, per ml at day 7, and 4.3 x lo3 TCID,, at day 11). The results suggest that HAV persists longer in mussels with low filtration ac- tivity.

In a further experiment, mussels were kept under optimal filtration conditions with a daily change of wa- ter. A homogenate of the filtration apparatus was made on day 1, 3, and 7. HAV concentration dropped from 6.3 x lo4 a t day 1, and 1 x lo4 at day 3, to 2 x lo2 TCID,, per ml at day 7. On day 3 the HAV concentra- tion in the hepatopancreas was slightly higher (5 x lo4 TCID,, per ml), and in the gonads equal to the concen-

Concentration of HAV in Mussels 177

TABLE I. Concentration of Infectious Hepatitis A Virus and Poliovirus as Detected by Tissue Culture Isolation Technique in Homogenates of Mytilus chilensis

at Different Exposure Times After Addition of Viruses to 1,000 ml Water of the Cultivation Systems Containing 6 Mussels Each*

Concentration of viruses Poliovirus Hepatitis A Virus

Inoculum (TCID50 in 1 liter water) Mussel extract (TCID5g per ml)

9.6 x 109

3.2 X lo6

2.0 x 1 0 7

3 hours after inoculation 3.2 x 107 3.5 x 105 1.7 x 105

3 days after inoculation 5.0 x 103 4.6 x 105 7 days after inoculation <3.0 X lo2 3.5 x 104

1 hour after inoculation 5.0 x 107 Not investigated

1 day after inoculation

*Mussels were kept under optimal filtration conditions (temperature 12OC, salinity of sea water 3%, daily change of water, feeding with 2 X lo7 Dunaliella marina twice a day, photoperiod 12 hours, continuous aeration).

tration found in the filtration apparatus (1 x lo4 TCID,, per ml). These results suggest that under opti- mal filtration conditions HAV will be eliminated faster from the filter apparatus than from the hepatopan- creas.

Comparison of Persistence of HAV and of Poliovirus in Mytilus chilensis

Twenty milliliters of tissue culture grown HAV (lo6 TCID,, per ml) or 3 ml of tissue culture grown poliovi- rus type 1 (3.2 x lo9 TCIDBo per ml of strain Mahoney), respectively, was added to two aquariums containing 6 mussels each. Aquariums were kept under optimal fil- tration condition for 7 days. The HAV concentration in mussel extract decreased only 10-fold in 7 days (from 3.5 x lo5 after 3 hours to 3.5 x lo4 TCID,, per ml after 7 days; see Table I). Poliovirus having an initial higher concentration in mussel extracts (5 x lo7 TCID,, per ml after 1 hour, and 3.2 x lo7 after 3 hours) was unde- tectable after 7 days (less than 3 x 10' TCIDSO per ml). Therefore, elimination of HAV from Mytilus chilensis is slower than elimination of poliovirus.

Concentration of HAV From Fecally Contaminated River Water in Mussels

Fecally contaminated river water was collected daily at the four locations indicated in Figure 1, and added daily to four shellfish cultivation systems containing two mussels each. Two hundred and thirty milliliters of river water was mixed with 770 ml of sea water. To keep the salinity of the culture system at 3% as in the experiments before, the salinity of sea water used for mixing had been increased by partially freezing the water and removing the ice.

Twenty-four hours after addition of the water col- lected on day 1, an extract was made from one of the two mussels in each cultivation system. The extract of the second mussel was made 24 hours after addition of wa- ter collected at day 14. Tenfold dilutions of each extract were inoculated into four cultures of PLC/PRF 5 cells. Tissue cultures were tested for the presence of HAV antigen after 6 weeks.

Only mussels cultivated in river water collected at location I expressed HAV. After a single feeding with river water, HAV was detected in one of four tissue cultures inoculated with a lop3 dilution of the mussel homogenate. A cytopathic effect (probably caused by toxic substances or by other enteroviruses) was seen in tissue cultures inoculated with the 10-1 and dilu- tion of the homogenate.

After inoculation of the homogenate of the mussel exposed for 14 days to river water from location I, three out of four tissue cultures became HAV antigen posi- tive at dilution of lop2, and two out of four cultures became positive at dilution of lop3. A cytopathic effect of the homogenate was present only at dilution of lo-.'.

For comparison, 2 ml of river water collected at the four locations on day 1 and 14 was inoculated directly into each of 4 cultures of PLCiPRF 5 cells. As expected, no culture became positive for hepatitis A antigen within 6 weeks of incubation. Therefore, concentration of HAV in shellfish may be used as a sensitive biologi- cal monitoring system to screen large amounts of water for the presence of infectious virus.

DISCUSSION Because of its physical and biochemical characteris-

tics, HAV has been classified as enterovirus 72. Entero- viruses are extremely stable under various environ- mental conditions and may survive for many weeks or even months in contaminated surface water, ground water, sea water, feces, sewage, sludge, and soil [Feachem et al., 19811. Several studies have shown that HAV is even more stable than most other enteroviruses [for review see Frosner, 19911. The infectivity of HAV is virtually unchanged for 4 weeks a t room temperature, and complete inactivation is only achieved a t tempera- tures above 60°C.

Filter feeding bivalve molluscs (mussels, oysters, clams) do not permit enterovirus replication, but are known to concentrate these viruses from surrounding water and to harbor viruses for a long period [Metcalf and Stiles, 1965; Metcalf et al., 1979; Tierney et al.,

178 Enriquez et al.

uptake of HAV. Elimination of HAV already present in mussels was also slower at low filtration activity: HAV could still be detected in mussels after 11 days. This last result is of special importance for depuration of commercially collected shellfish as it is performed in several countries [Mele et al., 19891. Water used for depuration should have suitable temperature and sa- linity, and should contain enough food to induce good filtration activity of shellfish.

HAV was demonstrated by virus isolation in extracts of mussels cultivated in large volumes (230 ml added daily) of fecally contaminated river water. Only water collected near the sewage outlet of two hospitals treat- ing hepatitis A patients caused positive isolation re- sults. As expected, direct inoculation of 2 ml of the same water specimens into each of four cultures of PLClPRF 5 cells revealed no HAV isolates. Therefore, the ability of shellfish to concentrate HAV from the surrounding water can be used as a sensitive biological monitoring system for detection of low level HAV contamination of river water, and possibly also for other enteroviruses.

ACKNOWLEDGMENTS This study was supported by Grant 1/62 668 of the

Volkswagen-Stiftung, Hannover, Germany. We thank Mrs. Marion Motz and Mrs. Sieglinde Blassmann for excellent technical assistance, and Dr. Reinhard Boh- mer for reviewing the manuscript.

19821. Persons ingesting raw or partially cooked (steamed) bivalve molluscs have been shown to be at great risk of acquiring viral hepatitis A [Portnoy et al., 1975; Dienstag et al., 1976; Yao, 19901. In many coun- tries, consumption of shellfish is a n important mode of transmission of hepatitis A. To control the disease, data on concentration and persistence of HAV in shellfish are needed, and methods for depuration of shellfish should be evaluated.

Most experimental data on the accumulation of en- teroviruses in shellfish have been obtained from oys- ters using poliovirus [for review see Gerba and Goyall, 1978; Feachem et al., 19811. Because isolation tech- niques for HAV in tissue culture are now available, concentration and persistence of this virus in Mytilus chilensis could be evaluated in this study. This mussel is frequently eaten in Chile, while other species of the genus Mytilus are consumed worldwide (Mytilus gallo- prouincialis, Mytilus edulis, Mytilus platensis).

Concentration and persistence of viruses are influ- enced by filtration activity of bivalve molluscs. Filtra- tion activity is largely dependent on environmental fac- tors such as temperature, salinity, and concentration of food particles of defined size in water. In most experi- ments in this study, optimal conditions for filtration activity of Mytilus chilensis were chosen: 12”C, salinity 3%, and feeding of 2-6 mussels present in 1 liter of cultivation fluid with 2 x lo7 cells of Dunaliella ma- rina twice a day [Navarro and Winter, 19821. Prior to usage of mussels in an experiment, they were kept un- der optimal filtration conditions in filtered sea water for a t least 10 days. This depuration time should be sufficient for cleansing out most bacteria and viruses which already may have been present in the mussels.

Our experiments suggest that under optimal filtra- tion conditions uptake of HAV by Mytilus chilensis oc- curs in less than 24 hours, and probably within 3 hours. HAV is concentrated about 100-fold relative to the sur- rounding water. These results are in agreement with data recorded for poliovirus in oysters. After 1 hour a 27-fold higher concentration was detected [Mitchell e t al., 19661. Other studies revealed 10- to 180-fold higher concentrations I Hoff and Becker, 19691.

As in another study with poliovirus in oysters [DiGi- rolamo et al., 19751, HAV was not only found in the filtration apparatus but also in high concentration in the digestive system (hepatopancreas). Therefore there is an uptake of HAV into the mussel tissue.

Under optimal filtration conditions, HAV could be detected in Mytilus chilensis €or about 7 days, suggest- ing a 1,000 to 10,000-fold reduction of virus concentra- tion during this time period. A 1,000-fold reduction of poliovirus was found in oysters already within 24 to 96 hours [Mitchell et al., 1966; Hoff and Becker, 19691. Direct comparison of elimination of HAV and poliovi- rus from Mytilus chilensis also confirmed that infec- tious HAV perists longer.

At low filtration activity due to not feeding the mus- sels with Dunaliella marina there was no detectable

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