Mussel homogenate was diluted 1:2 in glycine buffer (0·05 mol l−1, pH 9·00), homogenized, freeze–thawed and centrifuged at 8000 g for 15 min at 4 °C. Mussel extract supernatant fluid and the viral suspension were extracted twice with 30% chloroform. The interfaces were extracted with 500 μl cell culture medium (Eagle’s Minimum Essential Medium with Earle’s Salts (MEM); Imperial, UK). All the aqueous phases were collected together and, after centrifugation at 3000 g for 5 min, 100× antibiotics–antimycotic (Imperial) solution (1:100 v/v) was added to the supernatant fluid and stored at 4 °C overnight. The same amount of antibiotics–antimycotic (Imperial) solution was added and the sample maintained at 37 °C for 2 h.
Qualitative determination. Frp3 cells were grown with Eagle’s MEM supplemented with 1 mmol l−1 guanidine-HCl ( Siegl & Eggers 1982). Mussel extract (1 ml) and viral suspension extract (1 ml) were used to infect a 3-d Frp3 cellular monolayer. The cells were incubated for 2 weeks and evaluated for cytopathic effects. After the incubation period, all flasks (positive and negative for cytopathic effects) were frozen and thawed three times. After centrifugation at low speed (2000 g for 5 min), supernatant fluids were used for reverse transcriptase-polymerase chain reaction (RT-PCR) to confirm the presence of virus.
Samples which did not have a cytopathic effect on cells during the 15-d observation period, but were positive by RT-PCR, were used for a second infection. Thus, cellular suspension (1 ml) was placed in another flask containing Frp3 cells and incubated for an additional 15 d. Reverse transcriptase-PCR was performed to confirm the eventual observed cytopathic effect.
Samples positive for the presence of HAV by qualitative analysis, after the first 15 d of incubation, were subjected to quantitative determination.
Quantitative determination. The HAV titre in mussel extracts and in the viral suspension was measured on Frp3 cells. The infectivity titre was determined as TCID50 ml−1, using 24-well tissue culture plates and 100 ml of the inoculum ( Franco et al. 1990 ).
Four replicates were considered for each dilution. Results were confirmed by RT-PCR.
Reverse transcriptase-polymerase chain reaction. Hepatitis A virus primers were selected from the published sequences of the highly conserved VP2 and VP4 capsid region ( Beneduce et al. 1995 ) and checked for cross-reactivity with other enteric viruses by computer analysis (Advanced Blast Program, National Center for Biotechnology Information, Bethesda, MD, USA). The primer sequences are reported in Table 1.
Table 1. Hepatitis A virus primer sequences
RNA was extracted and purified using 334 μl HAV-infected cell lysate of each well and a CsCl (5·7 mol l−1) cushion, as described by Afzal & Minor (1994). The dried pellet was resuspended in 84 μl RT reaction buffer containing 20 mmol l−1 Tris-HCl (pH 8·4), 75 mmol l−1 KCl, 2·5 mmol l−1 MgCl2, 0·25 mmol l−1 of each deoxynucleoside triphosphate and 100 pmol primer 2 antisense. Twenty units RNasin (Promega, Madison, USA) and 1·25 U AMV reverse transcriptase (Promega) were added. The mixture was incubated at 42 °C for 50 min. The reaction was terminated by heating the mixture at 95 °C for 3 min. Primer 1 sense (100 pmol), 2·5 U Taq DNA polymerase (Promega) and diethyl pirocarbonate-treated water to a final volume of 100 μl were added.
The amplification was carried out for 30 cycles, each consisting of 25 s at 95 °C, 30 s at 37 °C and 1 min at 72 °C, followed by 5 min of final extension at 70 °C. The amplified product was analysed by agarose gel electrophoresis (2% agarose; Kodak, New Haven, USA) in the presence of 0·5 g ml−1 ethidium bromide and visualized under u.v. light.