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SUMMARY INTRO METHODS RESULTS DISCUSSION FOOTNOTES REFS
First posted online 13 September 2001 FULL-LENGTH ARTICLE
Rec 11 June 2001; Acc 31 August 2001 DOI: 10.1099/vir.0.17915-0

Genetic analysis of hepatitis A virus strains recovered from the environment and from patients with acute hepatitis

Sonia Pina,1 Maria Buti,2 Rosend Jardí,3 Pilar Clemente-Casares,1 Joan Jofre1 and Rosina Girones1

1 Department of Microbiology, University of Barcelona, Avd. Diagonal 645, 08028 Barcelona, Spain
2,3 Liver Unit2 and Department of Biochemistry3, Hospital General Universitario Valle Hebron, 08035 Barcelona, Spain

The molecular epidemiology of hepatitis A virus (HAV) was studied by analysing HAV strains recovered from environmental water samples over a 7 year period and strains recovered from patients with acute hepatitis over a 5 year period. A total of 54 samples of raw domestic sewage and 66 samples of river water were collected. HAV particles were concentrated and detected by nested RT–PCR. HAV infection in patients with acute hepatitis was serologically diagnosed in 26 of 74 serum samples, which were also analysed by nested RT–PCR. HAV RNA was detected in 57.4 % of sewage samples, 39.2 % of Llobregat river water samples, 20 % of Ter river water samples and 61.6 % of serum samples. The HAV genomes detected were characterized further by directly sequencing a region of the 5´ non-translated region, the VP1/2A junction region and, in some samples, the 2B region. Results showed a 95 % prevalence of genotype I, with nearly 50 % being either subgenotype IA or subgenotype IB. Various strains were found simultaneously in both environmental and clinical samples. These strains were closely related to those described in distant geographical areas. Genotype IIIA was also found in 5 % of sewage samples and in 12.5 % of serum samples. Strains belonging to a common endemic genotype were not identified. The abundance of HAV in the environment produces a situation of sanitary risk, especially considering the low prevalence of antibodies in the young population.


Introduction

Hepatitis A virus (HAV) has been classified as the only member of the genus Hepatovirus within the family Picornaviridae. As in other picornaviruses, the large open reading frame present in the HAV genome may be divided into three functional regions, termed P1, P2 and P3 (Rueckert & Wimmer, 1984). The P1 genomic region encodes the capsid polypeptides VP1, VP2, VP3 and VP4, and the P2 and P3 regions encode the non-structural polypeptides necessary for virus replication. The lack of antigenic diversity in HAV strains is related to a high degree of conservation in the amino acid sequences of the capsid proteins (Ticehurst et al., 1989).

HAV is an enterically transmitted picornavirus that causes acute hepatitis in humans and some primates. Transmission of the virus by the faecal–oral route results in the widespread endemic asymptomatic infection of infants and children in developing countries. Exposure of non-immune adolescent and adult populations within industrialized regions of the world results in clinical disease. HAV has a single antigenic serotype and a single infection is considered to confer lifelong immunity (Gellis et al., 1945; Lemon & Binn, 1983; Neefe et al., 1964; Rakela et al., 1976). The limited antigenic variability of HAV has precluded the use of serological approaches to differentiate precisely the sources of HAV infection in various epidemiologically defined settings of endemic and epidemic disease. Comparison of nucleotide sequences within a limited region of the genome is an alternative approach to define the genetic relatedness of different strains. Epidemiologically significant genomic analysis requires the study of the wild-type virus to circumvent any genetic changes associated with cell culture or animal adaptation. The use of nested PCR amplification and partial sequencing described here to study HAV strains detected in raw sewage, in other environmental samples and in clinical samples provides a framework for improving molecular epidemiology studies of HAV infection in a population.

Comparative studies of the nucleotide sequences of different HAV strains have suggested that sequence relatedness can be correlated with the geographical origin of the virus (Jansen et al., 1990; Robertson et al., 1991). Analysis of sequence data derived from the VP1/2A junction region allowed the description of seven distinct genotypes of HAV (Robertson et al., 1992).

Here, we depict HAV strains, identified in Barcelona (Spain), that have been recovered from both environmental water samples analysed over a 7 year period and clinical cases of acute hepatitis over a 5 year period. The study of these HAV strains will be useful for the design of improved strategies for the control of HAV infections.

Methods

Water samples. A total of 54 raw domestic sewage samples was collected from the sewage network of Barcelona (Spain) from 1994 to 2000 (100 % positive for faecal coliforms, with a mean concentration of 1.7x106 c.f.u./100 ml). Samples were taken at the entry of a treatment plant receiving sewage from a population of about 1.8 million people. Each sample was harvested in a sterile 500 ml polyethylene container and kept at 4 °C for less than 8 h until the virus particles were concentrated. The method applied for the recovery of virus particles from sewage samples was chosen on the basis of previous studies (Puig et al., 1994; Pina et al., 1998). Briefly, samples of 40 ml were ultracentrifuged at 229600 g for 1 h at 4 °C to sediment the virus particles together with any suspended material. Viruses retained in the sediment were eluted by mixing with 4 ml 0.25 M glycine buffer (pH 9.5) on ice for 30 min. The suspended solids were then removed by centrifugation at 12000 g for 15 min after the addition of 4 ml 2x PBS. Viruses in the supernatant were pelleted by ultracentrifugation at 229600 g for 1 h at 4 °C, resuspended in 0.1 ml PBS and stored at –80 ° C.

Water samples (n=56) from the Llobregat (50–250 l depending on the turbidity, 103–104 c.f.u./100 ml faecal coliforms) and Ter (n=10) (250 l, <10 c.f.u./100 ml faecal coliforms) rivers were collected from 1994 to 1998. The Llobregat and Ter rivers are located south and north of Barcelona, respectively. Both rivers receive large quantities of urban and industrial raw waste water, which contribute a high level of chemical and faecal contamination. Samples were concentrated primarily in situ using positively charged filters (Zeta-plus, CUNO) (Sobsey & Jones, 1979; Sobsey & Glass, 1980). Filter cartridges were transported to the laboratory and immersed in tryptone soy broth. The viruses retained were eluted with 1000 ml of 0.25 M glycine buffer (pH 9.5–10.5), concentrated by organic flocculation with 3 % beef extract (pH 3.5–4.0) (Becton Dickinson) and recovered in 50 ml PBS (APHA, 1995). Finally, virus particles were concentrated by ultracentrifugation, according to the method described for the sewage samples.

Human serum samples. Serum samples (n=74) from patients with acute hepatitis were randomly selected. Acute viral hepatitis was defined by the presence of specific clinical symptoms, which are as follows: serum alanine aminotransferase levels 10-fold higher than the normal upper values and exclusion of other causes of hepatocellular injury, including medications, alcohol, congestive heart failure, biliary disease, autoimmune hepatitis and other infections (e.g. cytomegalovirus and Epstein–Barr virus diseases). All serum samples were collected between October 1989 and December 2000 at the Hospital General Valle Hebron (located in Barcelona, Spain) from patients with symptoms of acute hepatitis. Samples were collected during the first month after the presentation of symptoms. Commercially available ELISA kits (Abbott) were used for the detection of IgM anti-HAV, HBV surface antigen, IgM anti-HBV, IgG anti-HEV and anti-HCV. HCV RNA was analysed by PCR (Amplicor HCV Monitor Test, version 2.0; Roche). Acute hepatitis A was diagnosed by the presence of IgM anti-HAV antibodies. Serum samples were stored at –80 °C until use for detecting genomic sequences.

Viruses. HAV strain pHM-175 was propagated in FRhK-4 cells growing in Eagle's minimal essential medium (MEM) (Auto-pow; ICN) containing 1.5 % sodium bicarbonate, 2 mM L-glutamine, non-essential amino acids, 100 U/ml penicillin, 100 µg/l streptomycin and 15 % foetal bovine serum. Virus suspensions were stored at –80 °C until use.

Nucleic acid extraction and enzymatic amplification. Viral nucleic acid from serum samples or virus stocks was extracted using guanidinium isothiocyanate, adsorbed onto silica particles and eluted in elution buffer containing 10 mM Tris–HCl (pH 8.0), 0.1 mM EDTA, 1 mM DTT and 0.24 U/µl ribonuclease inhibitor (Boom et al., 1990). RNA solutions were diluted 1:10 in elution buffer.

For the detection of RNA genomes, 5 µl aliquots of the extracted nucleic acid (corresponding to 0.5 and 5 µl serum, 0.2 and 2 ml sewage or 1 and 5 l river water), 1.5 mM MgCl2 and 1x PCR buffer II (Perkin-Elmer), containing 10 mM Tris–HCl (pH 8.3 at 25 °C), 50 mM KCl, 0.01 M DTT, 10 nmol of each dNTP and 25 pmol of the antisense primer (Table 1) in a total volume of 10 µl were used for reverse transcription. The reaction mixture was incubated at 95 °C for 5 min before the addition of 10 units of ribonuclease inhibitor (Applied Biosystems) and 50 units of reverse transcriptase from Moloney murine leukaemia virus (Applied Biosystems). After 30 min at 42 °C, the reaction was heated for 5 min at 95 °C.

Table 1. Oligonucleotide primers used for PCR amplification of HAV

The sequence position refers to the HM-175 strain of HAV (Cohen et al., 1987 b). For PCR, the melting temperature (Tm) was calculated according to the equation Tm=4x(number of pairs GC)+2x(number of pairs AT).

Region

Position

Name

Polarity

Tm (°C)

Product size (bp)

Sequence (5´ to 3´)*

5´ NTR

332–352

HAV1

Sense

64

368

TTGGAACGTCACCTTGCAGTG

680–700

HAV2

Antisense

64

  

CTGAGTACCTCAGAGGCAAAC

371–391

neHAV1

Sense

58

290

ATCTCTTTGATCTTCCACAAG

641–661

neHAV2

Antisense

64

  

GAACAGTCCAGCTGTCAATGG

332–351

HAV1d

Sense

62

376

TTGGRACGTCDCCTTGCAGT

689–708

HAV2d

Antisense

65

  

AAATGCCCCTGRGTACCTCAG

644–661

neHAV2d

Antisense

57

290

GWAMWGTCCAGCWDYHAATGG

VP1/2A

2906–2929

HHA1

Sense

59

532

TGCAAATTAYAAYCAYTCTGATGA

3416–3438

HHA2

Antisense

61

  

TTTCTGTCCATTTYTCATCATTC

2940–2960

HHA3

Sense

48

436

TTYAGTTGYTAYTTGTCTGT

3357–3376

HHA4

Antisense

55

  

TCAAGAGTCCACACACTTC

2B

3627–3646

2B1

Sense

58

343

GCAGATAGAATGCTTGGATT

3951–3970

2B2

Antisense

54

  

GAGTCCATYTGCCAATTAAA

3750–3769

2B3

Sense

59

220

TTTCAYCATTCTGTGACTG

* R=A or G; D=G, A or T; W=A or T; Y=C or T; M=A or C; H=A, T or C.

For a typical one-step PCR, a 10 µl sample of cDNA was used. Amplification was carried out in a 50 µl reaction mixture containing 10 mM Tris–HCl (pH 8.3 at 25 °C), 50 mM KCl, 1.5 mM MgCl2, 200 µM of each dNTP, 25 pmol of forward primer (Table 1) and 2 units of AmpliTaq DNA polymerase (Applied Biosystems). The amplification mixture was incubated in a GeneAmp 2400 PCR thermocycler (Applied Biosystems). The first cycle of denaturation was carried out for 3 min at 95 °C, followed by 30 cycles of denaturation at 95 °C for 60 s, annealing at 55 °C (for non-degenerate 5´ NTR primers), 42 °C (for degenerate VP1/2A primers) or 48 °C (for the primers of the 2B-region and the degenerate 5´ NTR primers) for 60 s and extension at 72 °C for 60 s. The final extension step was done at 72 °C for 5 min. Then, 1 µl (1/50) of the reaction mixture was added to a new batch of 50 µl PCR mixture, containing 25 pmol of each nested primer (Table 1) for a new PCR amplification cycle following the same procedure. PCR products were analysed by agarose gel electrophoresis using ethidium bromide as a stain.

To reduce the probability of sample contamination by amplified DNA molecules, standard precautions were applied in all manipulations. Separate areas were used for reagents, treatment of samples and manipulation of amplified samples. All samples were analysed twice in independent experiments and a negative control was added for each sample.

Sensitivity of detection. Sensitivity was evaluated by a limiting dilution experiment, as described previously (Puig et al., 1994; Pina et al., 2000). Serial 10-fold dilutions of HAV strain HM-175 RNA were tested by nested RT–PCR. Sewage samples were spiked with 2x103 genome equivalents per ml (GE/ml). One GE, as determined by RT–PCR, is defined as the number of genomes present in the highest serial dilution shown to be positive by RT–PCR. Virus particles were recovered by the method described above. Serial 10-fold dilutions of the RNA solution were reverse transcribed, amplified by the two-step PCR procedure described above and the limits of detection were then compared.

Sequencing and analysis of viral genomes. Nested PCR products were purified using the QIAquick PCR Purification kit (Qiagen). Both strands of purified DNA were sequenced with the ABI PRISM BigDye Terminator Cycle Sequencing Ready Reaction kit with AmpliTaq DNA polymerase (Applied Biosystems), according to the manufacturer's instructions. Results were checked using the ABI PRISM 3700 automated sequencer (Applied Biosystems). Sequences were then compared with the HAV sequences present in GenBank and EMBL using the BLAST N program, version 2.0.8, and aligned with the corresponding sequence fragments of the database using the CLUSTAL W program, version 1.8 (Thompson et al., 1994). Shading was carried out with the GeneDoc program, version 2.5.000.

The transition/transversion parameter was estimated from data sets using the PUZZLE program, version 4.0.2. Phylogenetic distances were determined by the maximum-likelihood method using the DNADIST program of the PHYLIP package, version 3.5c (Felsenstein, 1993). Phylograms were generated by the UPGMA algorithm using the NEIGHBOR program. The robustness of the grouping was determined by bootstrap resampling of the multiple sequence alignments (100 sets) with the programs SEQBOOT, DNADIST, NEIGHBOR and CONSENSE. Bootstrap values greater than 70 % provide statistical evidence of phylogenetic association (Felsenstein, 1985). The output graphics of the trees were created with the TREEVIEW package, version 1.5 (Page, 1996).

Nucleotide sequence accession numbers. GenBank accession numbers of the HAV strain sequences characterized previously and used for phylogenetic studies are shown in Table 2. The sequences reported in this paper have been deposited into GenBank under the following accession numbers: AF386798–AF386845 for the 5´ NTR sequences, AF386846–AF386889 for the VP1/2A junction sequences and AF386890–AF386911 for the 2B region sequences.

Table 2. HAV strains used for phylogenetic analysis

Strain

Genotype

GenBank accession no.

Reference

HM-175

IB

M14707

Cohen et al. (1987 b)

HM-175/24a

IB

M59810

Lemon et al. (1991)

HM-175/43c

IB

M59809

Lemon et al. (1991)

HM-175/18f

IB

M59808

Lemon et al. (1991)

HM-175/7MK5

IB

M16632

Cohen et al. (1987 a)

HAF-203

IB

AF268396

Unpublished

MBB

IB

M20273

Paul et al. (1987)

L-A-1

IB

AF314208

Unpublished

LA

IA

K02990

Najarian et al. (1985)

HAS-15

IA

X15464

Sverdlov et al. (1987)

GBM

IA

X75215

Graff et al. (1994)

CR326

IA

M10033

Linemeyer et al. (1985)

FG

IA

X83302

Beneduce et al. (1995)

FH1

IA

AB020567

Unpublished

FH2

IA

AB020568

Unpublished

FH3

IA

AB020569

Unpublished

AH1

IA

AB020564

Unpublished

AH2

IA

AB020565

Unpublished

AH3

IA

AB020566

Unpublished

CF53

II

M63025

Brown et al. (1991)

PA21

IIIA

M63026

Brown et al. (1991)

Nor-24

IIIA

AJ299467

Stene-Johansen et al. (1999)

Nor-20

IIIA

AJ299463

Stene-Johansen et al. (1999)

CY145

IV

M59286

Nainan et al. (1991)

AGM-27

V

D00924

Tsarev et al. (1991)

Results

Sensitivity

RNA extracted from the HAV strain HM-175 stock was amplified using primers specific for the 5´ NTR and VP1/2A region. Visible bands (obtained after agarose gel electrophoresis) were evident up to a 10–6 dilution after nested PCR, which corresponds to 1 GE. The estimated sensitivity for the detection of viral RNA in sewage samples spiked with HAV strain HM-175 was shown to be 10 GE, which is equivalent to the sensitivity of 1–10 virus particles, described previously when sewage samples spiked with enterovirus were analysed (Puig et al., 1994; Pina et al., 1998).

The sensitivity estimated for the detection of viral RNA in serum was shown in previous studies to be of the same order as the sensitivity observed for the virus stock when nested PCR amplification was applied (Pina et al., 2000); this demonstrated the absence of inhibitors of the amplification reaction.

Presence of HAV in environmental samples

HAV RNA was detected in 31 of 54 raw sewage samples tested (57.4 %) after nested RT–PCR using primers specific for the conserved 5´ NTR. RNA concentration was estimated by using a limiting dilution experiment and was found to range from 10 to 102 GE/ml. Samples with concentrations of RNA <10 GE/ml were deemed to be negative.

Of the 56 Llobregat river water samples, 22 (39.2 %) showed detectable levels of HAV, estimated to be between 10 and 102 GE/l. Viral RNA was also detected in 2 of 10 Ter river water samples tested (20 %) and the concentration was estimated to be 10 GE/l.

HAV in clinical samples

The diagnosis of acute HAV infection was confirmed by the presence of IgM anti-HAV in 26 of 74 serum samples taken from patients with acute hepatitis. Of the 26 patients, 16 (61.6 %) presented detectable levels of HAV RNA in the serum. These patients comprised 18 males and 8 females, with a mean age of 42 years (ranging from 24 to 79 years of age). Three patients were intravenous drug addicts and one had travelled to a developing country. Serum samples were obtained during the first week of symptoms in 10 cases, during the second week in 10 cases and between the third and the fourth week in six cases. All HAV RNA-positive samples were from patients whose serum was obtained between the first and the second week of presenting symptoms.

Sequence analysis of HAV RNA genomes

In order to characterize the HAV strains detected in environmental and clinical samples, nested PCR products from the 5´ NTR and the VP1/2A junction region were sequenced. Sequence analysis of 248 nt of the 290 bp nested PCR amplicon from the 5´ NTR of 33 environmental strains and 15 clinical strains showed a high degree of identity, ranging from 90 to 100 % among samples isolated between 1990 and 2000. Comparison of the nucleotide sequences with that of the control strain, HM-175, showed 91.5–99.1 % identity with the environmental strains and 90.7–98.4 % identity with the clinical strains (Tables 3 and 4). Comparative analysis with sequences from genetic databases indicated the presence of HAV variants that were related closely to genotypes IB (HM-175, MBB and L-A-1) and IA (GBM, LA, FG and FH), showing 94.7–100 % identity with the environmental samples and 97.5–100 % identity with the serum samples. Strains that cluster with strain PA21 (genotype IIIA) were identified in two sewage samples and in one serum sample, with 97–98 % identity (Tables 3 and 4). Two A residues at positions 551 and 591 in the 5´ NTR fragment are characteristic of the wild-type HM-175 strain. These nucleotides change to a G in cell culture-adapted strains after long-term passage (Lemon et al., 1991). All of the environmental and clinical strains detected contain the A at positions 551 and 591, as described for wild-type viruses (Lemon et al., 1991).

Table 3. Sequence comparison of the conserved 5´ NTR region (248 nt) of HAV strains detected in environmental samples from 1994 to 2000

 

Comparison with the control strain

Comparison with the identified strain

Sample*

No. of identical nucleotides

Identity (%)

No. of identical nucleotides

Identity (%)

Strain

Ll-05/09/94

246

99.1

248

100

HM-175, HAF-203

Ll-15/11/94

246

99.1

248

100

HM-175, HAF-203

SA-30/06/95

236

95.1

248

100

GBM, LA

SA-28/07/95

235

94.7

247

99.6

GBM

Ll-04/09/95

246

99.1

248

100

HM-175, HAF-203

Ll-18/12/95

246

99.1

248

100

HM-175, HAF-203

T-17/01/96

246

99.1

248

100

HM-175, HAF-203

Ll-24/02/97

244

98.3

246

99.1

MBB

Ll-21/05/97

235

94.7

246

99.1

FH3

T-18/06/97

238

95.9

240

96.7

MBB

Ll-06/08/97

246

99.1

248

100

HM-175, HAF-203

SA-07/10/97

238

95.9

245

98.7

FG

SA-06/11/97

238

95.9

245

98.7

FG

Ll-31/12/97

246

99.1

248

100

HM-175, HAF-203

SA-09/01/98

245

98.7

247

99.6

HM-175, HAF-203

SA-16/01/98

237

95.5

244

98.3

FG

SA-23/01/98

238

95.9

245

98.7

FG

SA-02/02/98

235

94.7

243

97.9

GBM, LA

SA-11/02/98

237

95.5

244

98.3

FG

Ll-25/02/98

243

97.9

247

99.5

MBB

SA-22/03/99

244

98.3

246

99.1

MBB

SA-26/03/99

241

97.1

243

97.9

GBM, LA†

SA-09/06/00

242

97.6

246

99.1

MBB

SA-23/06/00

242

97.6

238

95.9

GBM, LA†

SA-11/07/00

235

94.7

235

94.7

L-A-1†

SA-03/08/00

235

94.7

241

97.1

GBM, LA

SA-16/08/00

244

98.3

238

95.9

GBM, LA†

SA-01/09/00

236

95.1

242

97.5

GBM, LA

SA-05/10/00

234

94.4

246

99.1

GBM, LA

SA-26/10/00

244

98.3

228

91.9

PA21†

SA-06/11/00

244

98.3

246

99.1

HM-175, MBB

SA-04/12/00

227

91.5

243

98.3

PA21

SA-29/12/00

236

95.1

248

100

GBM, LA

* SA-dd/mm/yy, sewage sample; Ll-dd/mm/yy, Llobregat river water sample, T-dd/mm/yy, Ter river water sample.

† Data correspond to the strain identified according to hypervariable region analysis.

Table 4. Sequence comparison of the conserved 5´ NTR region (248 nt) of HAV strains detected in serum samples from 1990 to 2000
 

Comparison with the control strain

Comparison with the identified strain

Sample

No. of identical nucleotides

Identity (%)

No. of identical nucleotides

Identity (%)

Strain

HS-05/03/90

238

96.0

244

98.4

GBM, LA

HS-10/11/90

234

94.4

247

99.5

MBB

HS-17/01/91

237

95.6

243

97.9

GBM, LA

HS-27/02/91

243

98.0

247

99.5

MBB

HS-26/08/91

242

97.6

246

99.1

MBB

HS-17/09/91

236

95.2

248

100

GBM, LA

HS-25/09/91

241

97.2

245

98.7

MBB

HS-20/05/92

234

94.4

246

99.1

GBM, LA

HS-08/09/92

232

93.6

242

97.5

GBM, LA

HS-07/10/92

243

98.0

247

99.5

MBB

HS-06/04/99

244

98.4

246

99.1

HM-175, MBB

HS-27/04/00

233

93.9

245

98.7

GBM, LA

HS-16/10/00

234

94.4

246

99.1

GBM, LA

HS-13/11/00

225

90.7

242

98.3

PA21

HS-21/11/00

243

97.9

247

99.5

MBB

The VP1/2A junction region is recognized as one of the most variable regions of the HAV genome (Robertson et al., 1992) and was therefore chosen for genotyping and phylogenetic analysis. The sequence of 398 nt of the 436 bp nested PCR product from the VP1/2A region was determined for 27 strains detected in environmental water samples and in 16 clinical samples. Sequence comparison among the environmental and clinical samples showed 76–100 % identity and 76.8–99.2 % identity with the control strain, respectively. Comparative analysis with other isolates described previously confirms the presence of HAV strains belonging to genotypes IA and IB, with 93.7–100 % identity. Two of the sewage samples and two serum samples collected from October to December 2000 (SA-26/10/00, HS-13/11/00, SA-04/12/00 and HS-14/12/00) showed the presence of HAV strains related closely to strains PA21 and Nor-24 (genotype IIIA), with more than 99 % identity. The phylogenetic tree based on the alignment of the VP1/2A fragment is shown in Fig. 1.

Fig. 1Fig. 1. Phylogram depicting the relationship between the environmental and clinical HAV strains in relation to other isolates characterized previously over a 296 bp fragment of the VP1/2A junction region. Scale represents genetic distance. The internal node numbers represent bootstrap values (100 replicates) expressed as the percentage of all trees. Values greater than 70 % indicate statistically significant grouping (Felsenstein, 1985).


Point mutations were observed in the nucleotide sequence of the VP1/2A region of the environmental and clinical strains that differ from the control strain. The C at position 3018 of the control strain is changed to a T in all of the environmental and clinical strains. Moreover, all clinical isolates showed additional substitutions: the C at position 3002 in the HM-175 control strain is changed to a T in all strains detected in serum samples, the T at position 3248 is changed to an A, the G at position 3281 is changed to an A or a C, the G at position 3287 is changed to an A and the A at position 3353 is changed to a T. These mutations demonstrate the presence of wild-type strains in environmental water and clinical samples. Within the 2A region, the mutations described at positions 3248 and 3281 result in the substitution of the lysine and isoleucine residues at positions 47 and 58 to asparagine and methionine residues, respectively: these mutations are related to adaptation to cell culture growth (Lemon et al., 1991).

The results showed a similar prevalence of strains identified as genotypes IA and IB (55 and 40 %, respectively) in the environmental samples and 43.7 % of each subgenotype in serum samples. The strains related closely to genotype IIIA were identified in two environmental samples (5 %) and in two clinical samples (12.5 %).

We sequenced 182 nt of a 220 bp fragment within the 2B region in some of the environmental and clinical HAV strains. A mutation at position 3889 (C changed to a U), which has been described in cell culture-adapted strains, is responsible for an alanine to a valine substitution at amino acid position 72 of the 2B protein (Cohen et al., 1987 a; Emerson et al., 1993). All strain sequences showed a C in that position (data not shown), as described for wild-type strains, which eliminates the possibility of cross-contamination with laboratory strains.

Discussion

The study presented here provides a framework for global analysis of the HAV strains that circulate among the population in Barcelona and includes a description of the most prevalent HAV strains present in the environment and also those strains that resulted in cases of acute viral hepatitis. Results based on virus concentration from raw sewage or other environmental water samples, nested RT–PCR amplification and partial sequencing yields reliable information on the molecular epidemiology of HAV infection in an area located in the north-eastern region of Spain.

Sequence analysis of the environmental and clinical samples showed a high prevalence of genotype I strains. Genotype I comprise 80 % of HAV strains isolated worldwide, as described by Robertson et al. (1992). Genomic analysis shows that more than one HAV genotype could be identified within the same year in the same location; over a period of a few years, the most prevalent strains may differ. Genotypes IA and IB were found in the clinical samples collected from 1990 to 1992 at a proportion of 50 % each.

Genotype IB strains, related closely to strains HM-175, MBB and L-A-1, were identified in the environment throughout the period from 1994 to 2000. Genotype IA strains were also abundant and identified as GBM variants in 52.5 % of the isolates. LA- and HAS-15-related strains were the most prevalent during the year 2000 (in 5 of up to 12 isolates), together with MBB- and L-A-1-like strains (in 3 of up to 12 samples). Genotype IIIA variants were also detected in two samples.

A good concordance was observed between the strains detected in the environment and in clinical serum samples collected from patients with acute hepatitis during the year 2000, with genotypes IA, IB and IIIA circulating in the environment and among hepatitis patients. However, 100 % nucleotide identity in closely related strains detected in the clinical and environmental strains was never observed. This fact indicates that a high number of strains is currently circulating in the population, since it is accepted that, during infection, no sequence differences are usually detected, even between different patients involved in the same outbreak (Robertson et al., 1992).

Genotype III strains were detected in the environment and in hepatitis patients during the same period of time. This genotype has been demonstrated to be highly prevalent among intravenous drug users in Northern Europe (Robertson et al., 1992; Stene-Johansen et al., 1999). This is in agreement with the fact that one of the patients that presented a virus strain similar to strain Nor-24 belonged to that group, although in the second patient, no specific risk factor was identified.

HAV infection is considered to be endemic in the Mediterranean region and, as has been described previously in other countries, is characterized in Spain by diminishing HAV seroprevalence in the population (Papaevangelou, 1992; Dal-Ré et al., 2000; Lopalco et al., 2001). Within the specific area studied (Catalonia, Spain), a seroprevalence of 67.8 % in the general population has been documented; less than 5 % of a group of patients who were between 5 and 14 years of age were seropositive (Bruguera et al., 1999). Therefore, a large percentage of the Spanish population under 30 years of age is not protected against hepatitis. The results presented here show the circulation of several HAV strains identical to, or closely related to, strains isolated previously in highly divergent geographical areas worldwide. In this study, 79 HAV-positive samples collected over a period of 10 years were analysed. It was not possible to identify strains belonging to a common endemic genotype, as proposed by Robertson et al. (1992), suggesting the circulation of endemic strains in the USA between 1976 and 1990. Two hypotheses may explain this fact: (i) the higher frequency of travelling in the area and (ii) the higher prevalence of the infection in an endemic area, together with the fact that HAV has a high level of stability in the environment (Sobsey et al., 1988). During the year 2000, although the number of cases of hepatitis A has been estimated to be less than 15–30 per 100000 habitants, 80 % of urban sewage samples studied showed the presence of HAV. Since faecal shedding of HAV can, in some cases, last for months after the resolution of symptoms (Yotsuyanagi et al., 1996), such patients could be a source of further virus spreading within the community. It is important that this factor is considered as a possible reason for the abundance of HAV observed in the environment. Moreover, low levels of HAV multiplication in the intestinal epithelial cells has been suggested (Blank et al., 2000). This could represent a mechanism of virus amplification. Further monitoring will be needed to evaluate if there is a reduction in the presence of HAV in the environment over the next few years. Due to the low prevalence of antibodies in the young and the simultaneous abundance of HAV circulating in the environment, it may be important to consider the availability of a HAV vaccine in areas where sanitary is poor.

This work was partially supported by the research grants SGR/00023 from the Catalan Government and FIS 00/1123 from the 'Ministerio de Sanidad y Consumo' from the Spanish Government. Sonia Pina was in receipt of a fellowship from the Catalan Government. The authors thank the staff of the Servicios Científico-Técnicos of the University of Barcelona for their help in the sequencing of the PCR products. We also thank Drs S. U. Emerson and R. H. Purcell of the National Institutes of Health (NIAID, NIH, Bethesda, MD, USA) for their useful advice.

References

APHA (1995). Standard methods for the examination of water and wastewater, 19th edn. American Public Health Association, Washington DC, WA, USA.

Beneduce, F., Pisani, G., Divizia, M., Panà, A. & Morace, G. (1995). Complete nucleotide sequence of a cytopathic hepatitis A virus strain isolated in Italy. Virus Research 36, 299–309.

Blank, C. A., Anderson, D. A., Beard, M. & Lemon, S. M. (2000). Infection of polarized cultures of human intestinal epithelial cells with hepatitis A virus: vectorial release of progeny virions through apical cellular membranes. Journal of Virology 74, 6476–6484.

Boom, R., Sol, C. J. A., Salimans, M. M. M., Jansen, C. L., Wertheim-van Dillen, P. M. E. & van der Noordaa, J. (1990). Rapid and simple method for purification of nucleic acids. Journal of Clinical Microbiology 28, 495–503.

Brown, E. A., Day, S. P., Jansen, R. W. & Lemon, S. M. (1991). The 5´ nontranslated region of hepatitis A virus RNA: secondary structure and elements required for translation in vitro. Journal of Virology 65, 5828–5838.

Bruguera, M., Salleras, L., Plans, P., Vidal, J., Navas, E., Domínguez, A., Batalla, J., Taberner, J. L. & Espuñes, J. (1999). Changes in seroepidemiology of hepatitis A virus infection in Catalonia in the period 1989–1996. Implications for a new vaccination strategy. Medicina Clínica (Barcelona, Spain) 112, 406–408.

Cohen, J. I., Rosenblum, B., Ticehurst, J. R., Daemer, R. J., Feinstone, S. M. & Purcell, R. H. (1987 a). Complete nucleotide sequence of an attenuated hepatitis A virus: comparison with wild-type virus. Proceedings of the National Academy of Sciences, USA 84, 2497–2501.

Cohen, J. I., Ticehurst, R., Purcell, R. H., Bucker-White, A. & Baroudy, B. M. (1987 b). Complete nucleotide sequence of wild-type hepatitis A virus: comparison with different strains of hepatitis A virus and other picornaviruses. Journal of Virology 61, 50–59.

Dal-Ré, R., García-Corbeira, P. & García-de-Lomas, J. (2000). A large percentage of the Spanish population under 30 years of age is not protected against hepatitis A. Journal of Medical Virology 60, 363–366.

Emerson, S. U., Huang, Y. K. & Purcell, R. H. (1993). 2B and 2C mutations are essential but mutations throughout the genome of HAV contribute to adaptation to cell culture. Virology 184, 475–480.

Felsenstein, J. (1985). Confidence limits on phylogenies: an approach using the bootstrap. Evolution 39, 783–791.

Felsenstein, J. (1993). PHYLIP: Phylogeny Inference Package, version 3.5c. Department of Genetics, University of Washington, Seattle, WA, USA.

Gellis, S. S., Stokes, J., Brother, G. M., Hall, W. M., Gilmore, H. R., Beyer, E. & Morrisey, R. A. (1945). The use of human immune serum globulin (gamma globulin) in infectious (epidemic) hepatitis in the Mediterranean theatre of operations. Journal of the American Medical Association 128, 1062–1063.

Graff, J., Normann, A., Feinstone, S. M. & Flehmig, B. (1994). Nucleotide sequence of wild-type hepatitis A virus GBM in comparison with two cell culture-adapted variants. Journal of Virology 68, 548–554.

Jansen, R. W., Siegl, G. & Lemon, S. M. (1990). Molecular epidemiology of human hepatitis A virus defined by an antigen-capture polymerase chain reaction method. Proceedings of the National Academy of Sciences, USA 87, 2867–2871.

Lemon, S. M. & Binn, L. N. (1983). Antigenic relatedness of two strains of hepatitis A virus determined by cross-neutralization. Infection and Immunity 42, 418–420.

Lemon, S. M., Murphy, P. C., Shields, P. A., Ping, L. H., Feinstone, S. M., Cromeans, T. & Jansen, R. W. (1991). Antigenic and genetic variation in cytopathic hepatitis A virus variants arising during persistent infection: evidence for genetic recombination. Journal of Virology 65, 2056–2065.

Linemeyer, D. L., Menke, J. G., Martin-Gallardo, A., Hughes, J. V., Young, A. & Mitra, S. W. (1985). Molecular cloning and partial sequencing of hepatitis A viral cDNA. Journal of Virology 54, 247–255.

Lopalco, P. L., Salleras, L., Barbuti, S., Germinario, C., Bruguera, M., Buti, M. & Domínguez, A. (2001). Hepatitis A and B in children and adolescents: what can we learn from Puglia (Italy) and Catalonia (Spain)? Vaccine 19, 470–474.

Nainan, O. V., Margolis, H. S., Robertson, B. H., Balayan, M. & Brinton, M. A. (1991). Sequence analysis of a new hepatitis A virus naturally infecting cynomolgus macaques (Macaca fascicularis). Journal of General Virology 72, 1685–1689.

Najarian, R., Caput, D., Gee, W., Potter, S. J., Renard, A., Merryweather, J., Van Nest, G. & Dina, D. (1985). Primary structure and gene organization of human hepatitis A virus. Proceedings of the National Academy of Sciences, USA 82, 2627–2631.

Neefe, J. R., Gellis, S. S. & Stokes, J. (1964). Homologous serum hepatitis and infectious (epidemic) hepatitis. Journal of Medical Virology 1, 3–22.

Page, R. (1996). TREEVIEW: an application to display phylogenetic trees on personal computers. Computer Applications in the Biosciences 12, 357–358.

Papaevangelou, G. (1992). Epidemiology of hepatitis A in Mediterranean countries. Vaccine 10 (Suppl. 1), S63–S66.

Paul, A. V., Tada, H., von der Helm, K., Wissel, T., Kiehn, R., Wimmer, E. & Deinhardt, F. (1987). The entire nucleotide sequence of the genome of human hepatitis A virus (isolate MBB). Virus Research 8, 153–171.

Pina, S., Puig, M., Lucena, F., Jofre, J. & Girones, R. (1998). Viral pollution in the environment and in shellfish: human adenovirus detected by PCR as an index of human viruses. Applied and Environmental Microbiology 64, 3376–3382.

Pina, S., Buti, M., Cotrina, M., Piella, J. & Girones, R. (2000). HEV identified in serum from humans with acute hepatitis and in sewage of animal origin in Spain. Journal of Hepatology 33, 826–833.

Puig, M., Jofre, J., Lucena, F., Allard, A., Wadell, G. & Girones, R. (1994). Detection of adenoviruses and enteroviruses in polluted waters by nested PCR amplification. Applied and Environmental Microbiology 60, 2963–2970.

Rakela, J., Fay, O. H., Stevenson, D., Gordon, I. & Mosley, J. W. (1976). Similarities of two hepatitis A virus strains. Bulletin of the World Health Organization 54, 561–564.

Robertson, B. H., Khanna, B., Nainan, O. V. & Margolis, H. S. (1991). Epidemiologic patterns of wild-type hepatitis A virus determined by genetic variation. Journal of Infectious Diseases 163, 286–292.

Robertson, B. H., Jansen, R. W., Khanna, B., Totsuka, A., Nainan, O. V., Siegl, G., Widell, A., Margolis, H. S., Isomura, S., Ito, K., Ishizu, T., Moritsugu, Y. & Lemon, S. M. (1992). Genetic relatedness of hepatitis A virus strains recovered from different geographical regions. Journal of General Virology 73, 1365–1377.

Rueckert, R. R. & Wimmer, E. (1984). Systematic nomenclature of picornavirus proteins. Journal of Virology 50, 957–959.

Sobsey, M. D. & Glass, J. S. (1980). Poliovirus concentration from tap water with electropositive adsorbent filters. Applied and Environmental Microbiology 40, 201–210.

Sobsey, M. D. & Jones, B. L. (1979). Concentration of poliovirus from tap water using positively charged microporous filters. Applied and Environmental Microbiology 37, 588–595.

Sobsey, M. D., Shields, P. A., Hauchman, F. S., Davis, A. L., Rullman, V. A. & Bosch, A. (1988). Survival and persistence of hepatitis A virus in environmental samples. In Viral Hepatitis and Liver Diseases, pp. 121–124. Edited by A. J. Zuckerman. New York: Alan R. Liss.

Stene-Johansen, K., Skaug, K. & Blystad, H. (1999). Surveillance of hepatitis A by molecular epidemiologic studies. Tidsskrift for den Norske Laegeforening 119, 3725–3729.

Sverdlov, S. D., Tsarev, S. A., Markova, S. V., Vasilenko, S. K., Chizhikov, V. E., Petrov, N. A., Kusov, Y. Y., Natashenko, T. A. & Balayan, M. S. (1987). Cloning and expression of hepatitis A virus genome in E. coli cells. Molekulyarnaya Genetika Mikrobiologiya i Virusologiya 6, 129–133.

Thompson, J. D., Higgins, D. G. & Gibson, T. J. (1994). CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, positions-specific gap penalties and weight matrix choice. Nucleic Acids Research 22, 4673–4680.

Ticehurst, J., Cohen, J. I., Feinstone, S. M., Purcell, R. H., Jansen, R. W. & Lemon, S. M. (1989). Replication of hepatitis A virus: new ideas from studies with cloned cDNA. In Molecular Aspects of Picornaviral Infection and Detection, pp. 27–50. Edited by B. L. Semler & E. Ehrenfeld. Washington DC: American Society for Microbiology.

Tsarev, S. A., Emerson, S. U., Balayan, M. S., Ticehurst, J. & Purcell, R. H. (1991). Simian hepatitis A virus (HAV) strain AGM-27: comparison of genome structure and growth in cell culture with other HAV strains. Journal of General Virology 72, 1677–1683.

Yotsuyanagi, H., Koike, K., Yasuda, K., Moriya, K., Shintani, Y., Fujie, H., Kurokawa, K. & Lino, S. (1996). Prolonged fecal excretion of hepatitis A virus in adult patients with hepatitis A as determined by polymerase chain reaction. Hepatology 24, 10–13.


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