APPLIED ANDENVIRONMENTALMICROBIOLOGY, Nov. 1995, p. 3977–3980 Vol. 61, No. 11 0099-2240/95/$04.0010
Copyrightq1995, American Society for Microbiology
Direct Sequencing of Hepatitis A Virus Strains Isolated during an Epidemic in France
V. APAIRE-MARCHAIS,1B. H. ROBERTSON,2V. AUBINEAU-FERRE,1M. G. LE ROUX,3 F. LEVEQUE,4L. SCHWARTZBROD,5ANDS. BILLAUDEL1*
Laboratoire de Virologie, Institut de Biologie, 44035 Nantes Cedex 01,1Laboratoire de Ge´ne´tique Mole´culaire, Nantes,3Centre de Recherche du Service de Sante´ des Arme´es, Grenoble,4and De´partement de
Virologie, Faculte´ de Pharmacie de Nancy, Nancy,5France, and Hepatitis Branch, National Center for Infectious Diseases, Centers for Disease
Control and Prevention, Atlanta, Georgia2 Received 2 June 1995/Accepted 1 September 1995
Direct sequencing of PCR products was used to study the VP1 region of the hepatitis A virus (HAV) genome (position 2199 to 2356) of nine strains isolated from human stools collected during a hepatitis A epidemic (western France, 1992), three strains from environmental samples (1990, 1991, and 1992), and two HAV cell culture isolates (the French strain CF53/Lyon and strain CLF). These viruses differed from CF53/Lyon (genotype I) by between 1 and 10.3%, and results indicated the existence of two groups of strains belonging to two different subgenotypes (IA and IB). With this sequencing technique it was possible to monitor the epidemiology of HAV and study its relations.
Different hepatitis A virus (HAV) strains worldwide, cor- responding to a single serotype, have been classified into dif- ferent genotypes. This classification is based upon reverse transcriptase PCR amplification of the genome followed by sequence analysis. Regions that have been sequenced for these classifications include the C-terminal regions of VP3 (12020, 22211) and VP1 (12984,23265) (5), the VP1 amino-terminal region (12193,22389) (12), and the VP1/P2A junction (12984, 23285;12984,23265;12949,23285;12934,23285;12799, 23273) (7, 13). The last region was chosen for analysis of a se- quence of 168 bp in 152 strains from different countries, re- sulting in the definition of seven distinct genotypes (four hu- man [I, II, III, VII] and three simian [IV, V, VI]) differing in homology by 15 to 25%. Most of the human strains studied have been classified as genotype I, while the second most prevalent human group was genotype III. The strains included in these studies come mainly from the United States, Sweden, and Japan, whereas Africa, south Asia (India), and South America, where hepatitis A is highly endemic, are inadequately represented. Most of the strains isolated in Europe come from Sweden and belong to genotypes I and III (13).
A study was carried out by the local health authority (Di- rection De´partementale des Affaires Sanitaires et Sociales of the Loire-Atlantique) between January 1990 and September 1991 to evaluate the presence of HAV and enteroviruses in shellfish harvested in beach gathering areas by tourists for their own consumption and where people go to swim and play in the water. The HAV genome has been detected by molecular hybridization in numerous cockle and mussel samples (6). Be- tween December 1991 and March 1992, a HAV epidemic oc- curred in western France (Loire-Atlantique and Morbihan) in a region where shellfish farming is highly developed. Around 800 cases were recorded, with the peak of hepatitis A cases occurring in February, and shellfish consumption was consid- ered the likely cause.
During the epidemic, stools of HAV-infected patients were collected between January 1992 and March 1992 from different
locations in France, and the virus genome was detected by a reverse transcriptase PCR technique developed in our labora- tory (2). Epidemiological data indicated that patients con- sumed different species of shellfish. After the epidemic the Ministe`re de la Sante´ et de l’Action Humanitaire decided to control two areas on the Atlantic coast, and from May to August 1992, shellfish were collected (4). In addition, shellfish samples from 1990, 1991, and 1992, as well as stools from 1992, were available, and we used PCR products to sequence the VP1 region (2199 to 2356) of these different strains. In the study presented here, we analyzed the sequences obtained and compared them with the sequence of the French CF53/Lyon strain grown in PLC/PRF/5 cells and with MBB, HM175, LA, and CLF strains.
* Corresponding author.
FIG. 1. Reverse transcriptase seminested PCR. Results obtained in UV light after polyacrylamide gel electrophoresis. Lanes: A, positive control; B to D, negative control; F and G, samples 17 and 18; H, negative sample; I to L, samples 14, 6, 24, 2.
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MATERIALS AND METHODS
Virus strains.The CF-53/Lyon HAV strain (passage 28, titer 10750% tissue culture infectious doses per ml), donated by R. Deloince (Centre de Recherche du Service de Sante´ des Arme´es, Grenoble, France), and the CLF HAV strain (passage 10, titer 107to 10850% tissue culture infectious doses per ml), provided by G. Siegl (Institut fu¨r Klinische Mikrobiologie und Immunologie, St. Gallen, Switzerland), were used as positive controls for PCR and as references for sequencing. Only the CLF HAV strain was passaged four times in MRC-5 cells in our laboratory. Poliovirus type 1 (Sabin strain), provided by M. Aymard (Enterovirus Reference Center, Lyon, France), was used as the specificity con- trol.
Sequences of strains HM175 (genotype IB) (3), MBB (genotype IB) (10), LA (genotype IA) (8), and SLF88 (genotype VII) were used for computer analysis of sequence comparison (Table 1). Strain CF53/Berne (passage 6) was donated by R. Deloince to G. Siegl, passaged four times in PLC/PRC/5 and MRC-5 cells, and then sequenced in the region VP1/P2A by S. Lemon (5). This CF53/Berne strain was classified as genotype II.
Stool and shellfish samples.The stool samples used as a source for amplifi- cation of wild-type viral nucleic acid are listed in Table 2. HAV was detected in nine stool samples from patients with hepatitis A, which were collected by private regional laboratories from January 1992 to March 1992, and in three shellfish (cockle) samples (C4, C2, and C18) collected from the same area near the Loire River estuary (La Baule) before and after the epidemic (30 May 1990, 26 July 1991, and 20 July 1992, respectively).
Oligonucleotide primers and probe.The primers used for the first amplifica- tion reaction, HAV 2389–2414 and HAV 2169–2192, were described by Robert- son (11). For seminested PCR, we chose an antisense primer, located between nucleotides 2358 and 2377 (59TCC CAA TTG TTG GAT AGC 39), and the same positive-sense primer (HAV 2167–2192).
Sequencing was performed by using HAV 2167 or HAV 2358 on seminested PCR products. The sequence of the positive-sense primer HAV 2167 was derived from nucleotides 2167 to 2192 (based on the HM175 nucleotide numbering scheme of Cohen et al. [3]) within the C terminus of the VP3 genome region, and the sequence of negative-sense primer HAV 2358 was derived from nucleotides 2358 to 2377. An internal digoxigenin-labeled oligonucleotide probe (59TCC TCA ACA GTT TCT ACA GA 39, nucleotide 2232 to 2251, HM175) was used for hybridization analysis (1).
Preparation of RNA HAV, reverse transcription, and amplifications.Extrac- tion of HAV RNA from cell culture, stool, or shellfish samples was performed by previously described methods (2, 6). RNA was then reverse transcribed in cDNA using reverse transcriptase (MuMLV, Stratagene) prior to PCR amplification (seminested PCR) (2).
Purification and sequencing of double-stranded PCR products.To provide a high-quality template for direct sequencing of PCR-amplified DNA, 100ml of PCR products was purified by centrifugation in a Microcon concentrator (Mi-
crocon 100; Grace Amicon) in order to separate double-stranded cDNA prod- ucts from extraneous amplification products. One-tenth of the Microcon-purified PCR product was then evaluated by electrophoresis in a 9% polyacrylamide gel.
Purified PCR products were sequenced by the dideoxy method (Sanger) by using the Circumvent Thermal Cycle dideoxy DNA sequencing kit with vent R (exo-) DNA polymerase (New England Biolabs). DNA (0.01 pmol of double- stranded DNA) and primer A or C (1.2 pmol) were combined in 1.5ml of 103 Circumvent Sequencing buffer–1ml of 303Triton X-100 solution–distilled water to a total volume of 12 ml. On ice, 1ml of35S-dATP (Amersham) (.1,000 ci/mmol) and 2 U of vent R (exo-) DNA polymerase were added to the solution, and 3.2ml of this reaction mixture was immediately added to 3ml of each dideoxy A, C, G, and T mixture. Each reaction was overlaid with mineral oil (Sigma), and the tubes were placed in a thermal cycler. After a 958C denaturation step (5 min), termination reactions were performed with 20 cycles (958C for 30 s, 558C for 30 s, and 758C for 30 s). Reactions were then stopped by the addition of 4ml of stop-loading dye solution beneath the mineral oil. After each mixture was heated at 958C for 5 min, 4ml was electrophoresed in 6% polyacrylamide–8 M urea gels (polyacrylamide [19:1, Bioprobe], urea [8 M, Sigma], TBE [13, Bioprobe]) in TBE 13buffer at 50 W and then exposed to film (Kodak) for 1 to 3 days.
Computer analysis of sequences.Nucleotide sequences were compared by using PC-Gene analysis software (Intelligenetics, Inc) and the Piling program (Genetics Computer Group, program manual for the GCG Package, Madison, Wis.).
RESULTS
Detection and hybridization analysis of the amplified VP1 genome region.Nine of the stools collected during the hepatitis A epidemic in 1992 were selected on the basis of the geo- graphic origin of the patient (no. 2, 6, 8, 10, 14, 16, 17, 18, and 24). The HAV genome for all of these stools was detected by seminested PCR and confirmed by hybridization with the spe- cific digoxigenin-labeled D oligoprobe (2). However, only the PCR results for samples 2, 6, 14, 17, 18, and 24 are presented in Fig. 1. An amplification reaction was also performed on
FIG. 2. Sequence obtained from PCR products for 148 nucleotides of the VP1 genome region. Consensus sequence is depicted on last line. Dashes indicate presence of the most common nucleotide at the particular position.
TABLE 1. Genotyped strains used during analyses
HAV strain Genotype Yr Location
CF53/Berne II 1979 France
CLF IB 1983 Switzerland
HM175 IB 1976 Australia
MBB IB 1978 North Africa
LA IA 1975 Los Angeles
SLF88 VII 1988 Sierra Leone
TABLE 2. Human samples from which HAV was isolated by PCR
Patient
no. Sexa Age
(yr)
Dateb
Location Fecal collection Serodiagnosis
16 F 12 92/02/27 92/02/20 Lorient
17 F .15 92/02/27 ND Lorient
18 F ,15 92/02/27 ND Lorient
14 M 40 92/02/27 92/02/17 Lorient
6 F 27 92/02/13 92/02/03 Nantes
24 M 30 92/03/11 92/02/08 Nantes
2 M 33 92/02/14 92/02/05 Nantes
8 M 9 92/02/20 92/02/20 Tours
10 M 9 92/02/20 92/02/20 Tours
aF, female; M, male.
bDate format: year/day/month. ND, not determined.
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three shellfish samples (C2, C4, and C18) containing the HAV genome detected by RNA-RNA molecular hybridization (6).
Analysis of VP1 sequence variation.We analyzed the nucle- otide sequence (148 bp) located in the 2199–2356 region of two HAV strains isolated in culture (CLF, CF53/Lyon) and of various strains isolated from human stools and shellfish in the environment. The sequences obtained (Fig. 2) were compared with reference strains LA (genotype IA), MBB and HM175 (genotype IB), and SLF88 (genotype VII).
Compared to each other, strains C2 and C18 were more closely related to each other (94% similarity). The sequence of strain C4 was more closely related to that of the genotype IA (Fig. 2 and 3) reference isolate LA, while strains C2 and C18 were more similar to genotype IB reference strains MBB and HM-175 (Fig. 2).
Stool samples from individuals in three separate regions of France were collected and evaluated during the winter of 1992.
The virus characterized in cases from Tours was genotype IA and differed by a single nucleotide from environmental strain C4 (Fig. 2) which had been collected from shellfish beds during the summer of 1990. Testing of the stool samples from Nantes and Lorient revealed that the viruses were genotype IB (Fig. 2 and 3) but were distinct from environmental strains C2 and
C18, collected from shellfish beds during the summer of 1991 and 1992.
During our study, the sequence of the VP1 region of strain CF53/Lyon was determined, and it was found to be genotype IB. Strains CF53/Lyon and CLF differed by a single nucleotide (Fig. 2) (98% homology), and the sequence of CLF was found to be identical to that of MBB. Previous data have indicated that cell-adapted MBB and CLF are the same virus, most probably because of cell culture cross-contamination (5, 13).
The CF53/Berne strain is genotype II, based on its sequence within the VP3 C terminus and the VP1/P2A junction, while CF53/Lyon is genotype IB. According to the classification of Robertson et al. (13), SLF88, the sole representative of geno- type VII, is the closest strain to CF53/Berne (genotype II), based on comparison of sequence within the VP1/P2A region.
As shown in Fig. 3, a graphic representation of the relationship between SLF88 and the strains we have sequenced confirms the classification in different genotypes.
Comparison of the amino acid sequence in VP1 (Fig. 4).The consensus amino acid sequences for the subgenotype IA and subgenotype IB are shown in Fig. 4. Beneath the consensus sequence is shown the derived amino acid sequence for the strains described in this study. All of the strains of subgenotype IA (C4, Tours 8, Tours 10) differ from subgenotype IB strains (C2, C18, 14, 24, 2, 16, 17, 18, 6) in that they have a Arg substitution at Lys-520 and a Lys substitution at Arg-528.
DISCUSSION
An epidemic in western France during the first three months of 1992 allowed us to collect stool samples, detect the HAV genome in some specimens, and sequence a portion of the viruses after PCR amplification. On the basis of the initial studies of Robertson (12), we began our work by sequencing the VP1 region.
We detected two groups of human strains based upon geo- graphical origin: strains 8 and 10 from Tours, a city in western France 200 km from Nantes, and strains 16, 17, and 18 from Lorient in western France, which were similar to strains 2, 24, and 6 from patients hospitalized in Nantes. Sample 14 from the Lorient epidemic area was positive in PCR and negative in hybridization after several controls. Analysis showed that there was a G in the place of an A (position 2237 in Fig. 2) in the sequence corresponding to the oligoprobe, whereas this se- quence was highly conserved in the other strains. Despite this mutation, sample 14 was related to other strains from Lorient.
During the epidemic, a survey was performed and 800 cases were recorded. To monitor the presence of HAV after the
FIG. 3. Genetic relatedness between various HAV French strains isolated from human stools and shellfish in the environment, HAV strains isolated in culture (CLF, CF53/Lyon), and HAV reference strains (LA, MBB, HM175, SLF88). The dendrogram illustrates a pairwise comparison of sequences within a 148-base segment in the VP1 amino-terminal region (2199–2356). The location of the node between strains indicates the approximate percentage nucleotide identity between sequences shown on the abscissa. Roman numerals designate the respective genotype groupings, whereas a and b designate subgenotypes.
FIG. 4. Comparison of the amino acid sequences in the VP1 amino-terminal region. Consensus amino acid sequences for the predominant subgenotypes Ia and Ib are shown. Dashes indicate conserved amino acids; differences are shown by the appropriate single-letter amino acid code.
FIG. 2—Continued.
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epidemic, the Ministe`re de la Sante´ et de l’Action Humanitaire controlled two areas on the Atlantic coast and shellfish samples were collected from May to August 1992. Shellfish collections had been already performed within these areas between Jan- uary 1990 and September 1991, but no samples were collected just before the 1992 epidemic. Environmental samples were available from an area near the Loire river estuary from 1990, 1991, and 1992, and we proceeded to determine the sequence of virus derived from these sources and to compare them with the human strains. Environmental strain C4 collected in 1990 was similar to strains isolated in Tours but notably different from C2 (1991) as well as from C18 (1992), which was collected after the epidemic.
Shellfish consumption was the most plausible explanation, given the food habits of the population involved and the oc- currence of the outbreak during the winter (9). However, di- rect connection between shellfish consumption and the viruses found in the cases of hepatitis A from the three regions was not established. The samples from Tours were collected from chil- dren living in a psychiatric institute. Cases of hepatitis A oc- curred during February 1992, but shellfish consumption was most unlikely to explain these cases. Consumption of shellfish (clams, oysters) before the onset of the symptoms was related only for samples from two patients (14 and 16) from Lorient.
To demonstrate a correlation between shellfish consumption and cases of hepatitis A, one would need to recover virus sequences from shellfish samples that had come from the col- lection beds at the appropriate harvest time and show that the viruses in the shellfish and the patient were identical, prefer- ably both in the VP1 amino acid terminus and within the VP1/P2A junction.
Our data indicate that both subgenotypes IA and IB are circulating within France, as they were found in both the shell- fish samples and in the patients who became ill during the winter/spring of 1992. The virus from the patients in Tours was subgenotype IA, similar to strains collected from North Amer- ica, China, and Russia, while the viruses from the Nantes and the Lorient patients were all subgenotype IB, which has been found in Australia, Europe, Japan, and South America.
The French strain CF53/Lyon, sequenced in our laboratory, differed from the 1992 wild-type epidemic strains by 1 to 10.3%
and was clearly genotype IB. In France, CF53/Lyon was prop- agated only in cell culture in R. Deloince’s laboratory prior to being sequenced. CF53/Berne (5, 13) was determined to be genotype II. This strain (passage 6) was donated from R. De- loince to G. Siegl, who passaged it four times prior to being sequenced by S. Lemon in the VP1/P2A junction. The discrep- ancy of genotypes between CF53/Lyon and CF53/Berne may be a result of the fact that both of the isolates have been subjected to cell culture, where cross-contamination easily oc- curs (10a), and has been observed in other strains (5, 13). The other possibility is PCR contamination. Separate PCR ampli- fications and sequencing reactions were performed, ruling out the possibility of misincorporation during reverse transcription and PCR amplification.
With the sequencing technique based on PCR products, it is possible to monitor the epidemiology of this virus and study its relations. Genotyping can be used to trace cases involving a common source of infection or serial transmission. Genotypes IA and IB are circulating within France. Are there genotypes within France? Epidemiological monitoring of HAV French strains is of considerable interest for environmental and hu- man strains and should be further developed.
ACKNOWLEDGMENTS
We acknowledge R. Deloince for critical reading of the manuscript and for kindly providing the HAV CF53 strain. Shellfish collection was carried out by the Direction De´partementale des Affaires Sanitaires et Sociales de Loire Atlantique, and we are grateful to J. Duchemin and M. A. Goraguer. We thank R. Breatnach for providing computer algorithms.
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