Genetic characterization of wild-type genotype VII hepatitis A virus

Karen Z. Ching1, Tatsunori Nakano1, Louisa E. Chapmanb,2, Austin Dembyc,2 and Betty H. Robertson1

Hepatitis Branch1 and Special Pathogens Branch2, Division of Viral and Rickettsial Diseases, National Center for Infectious Diseases, Centers for Disease Control and Prevention, 1600 Clifton Road NE A33, Atlanta, GA 30333, USA

Author for correspondence: Betty H. Robertson. Fax +1 404 639 1563. e-mail bjr1{at}cdc.gov


   Abstract
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Abstract
Introduction
Methods
Results
Discussion
References
 
The complete genome sequence of the only identified genotype VII hepatitis A virus (HAV), strain SLF88, was obtained from PCR amplicons generated by a modified long PCR approach. There was 90% nucleotide identity in the 5' untranslated region compared to other known HAV sequences. In the remainder of the genome containing the long open reading frame, there was about 85% nucleotide identity to human HAV genotypes IA and IB and 80% identity to simian HAV genotype V. Compared to HAV strain HM-175, the capsid amino acids were highly conserved, with only four homologous amino acid changes, while an increasing number of amino acid differences was seen in the P2 and P3 genome regions. While nucleotide variability within the three functional coding regions did not differ, the P3D region was found to have the largest number of amino acid changes compared to HM-175.


   Introduction
Top
Abstract
Introduction
Methods
Results
Discussion
References
 
Hepatitis A virus (HAV) is a 7·5 kb positive-stranded RNA virus belonging to the family Picornaviridae (Melnick, 1982 ; Gust et al., 1983 ) and has been designated a member of the genus Hepatovirus (Minor, 1991 ). As a picornavirus, the genome can be defined into four distinct regions. The 5' untranslated region (UTR) contains extensive secondary structure necessary for cap-independent translation. The P1 region encodes the structural polypeptides (VP1, VP2, VP3 and a putative VP4). The P2 and P3 regions encode the nonstructural proteins associated with replication; the function of three of these, P3A, P3C and P3D, have been identified as the VPg, the viral protease and the RNA-dependent RNA polymerase (Weitz et al., 1986 ; Cohen et al., 1987 ; Tesar et al., 1994 ; Jia et al., 1991 ).

Antibodies to human HAV cannot distinguish individual strains of HAV and only a single serotype of human HAV has been documented. However, genetic variants of HAV have been identified by sequencing selected, short genome regions, including the VP3 C terminus (Jansen et al., 1990 ), the VP1 amino terminus (Robertson et al., 1991 ) and the VP1/P2A junction region (Jansen et al., 1990 ; Robertson et al., 1992 ). Seven HAV genotypes have been defined based upon the sequence of the VP1/P2A junction region of a global collection of viruses (Robertson et al., 1992 ). Genotypes were defined by sequences that differed from each other in these regions by at least 15%; subgenotypes differed by 7·0–7·5%. These studies identified four genotypes (I, II, III and VII) associated with human HAV infections and three simian HAV strains (IV, V and VI). Genotypes IA and IB appear to be the HAV genotypes identified most frequently in North and South America, Europe, China and Japan (Robertson et al., 1992 ); African strains of HAV include strains of genotypes IA and IB identified in South Africa (Taylor, 1997 ) and isolated travel-associated cases from North Africa (genotype IB), Tunisia (genotype IB) and Nigeria (genotype IA) and a single representative of genotype VII from Sierra Leone (Robertson et al., 1992 ).

Complete genome information is available for a number of human genotype IA and IB HAV strains and for the simian HAV genotype V. However, complete genome information from the remaining human HAV genotypes (II, IIIA, IIIB and VII) and the simian HAV genotypes IV and VI is not available. These data, presented in these studies, characterized the complete genome sequence from the only virus representing human HAV genotype VII, designated SLF88. This virus was responsible for two epidemiologically linked fulminant hepatitis A cases and is the only identified strain from Sierra Leone.


   Methods
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Abstract
Introduction
Methods
Results
Discussion
References
 
{blacksquare} Processing of liver specimens and RNA extraction.
The liver from the index case was obtained at autopsy. A 20% (w/v) suspension was prepared by homogenization in 50 mM Tris–HCl and 100 mM NaCl, pH 7·5. Liver homogenates were freeze–thawed three times and NP-40 added to a final concentration of 1%. After 30 min on ice with gentle mixing every 10 min, nuclei and cell debris were pelleted by centrifugation at 12000 g for 10 min at 4 °C and aliquots stored at -70 °C. Viral RNA was isolated from 100 µl of this liver homogenate with Trizol (Life technologies), according to the manufacturer’s protocol, with the exception that repeated, gentle tube inversion to prevent RNA shearing was used instead of vortexing.

{blacksquare} cDNA synthesis.
The RNA pellet was resuspended in 38·5 µl of reverse transcription mixture containing 2·5 µl of 10 mM reverse primer stock solution (HAV3', Table 1; Tellier et al., 1996 ), 1 µl of random primer (Promega), 1 µl of 10 mM each of four dNTPs, 8 µl of 25 mM MgCl2 and 2 µl DMSO. The RNA–primer solution was incubated for 2 min at 65 °C and rapidly chilled on dry ice. AMV reverse transcriptase (25 U, 1 µl) (Boehringer Mannheim) and 1 U (0·5 µl) RNase ribonuclease inhibitor (Promega) were added and the samples incubated for 1 h at 42 °C. To inactivate the reverse transcriptase, the mixture was heated at 95 °C for 5 min and then chilled on ice.


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Table 1. Primers used for genome and large fragment amplification

 
{blacksquare} Long PCR.
PCRs were performed in thin-walled PCR tubes in a total volume of 50 µl containing 1 µl of 10 mM each of four dNTPs, 2 µl each of 10 mM positive- and negative-sense primer solutions, 5 µl 10xPCR reaction buffer [40 mM Tricine-Koit pH 9·2, 15 mM potassium acetate, 3·5 mM magnesium acetate and 375 µg/ml BSA (Clontech), 10 µl template cDNA, 1 µl Advantage DNA polymerase (Clontech) and 2 µl DMSO]. PCR was performed in a 9600 Thermal cycler (Applied Biosystems) using the following conditions: hot start at 95 °C for 1 min, denaturation at 96 °C for 35 s, annealing at 60 °C for 30 s, extension at 68 °C for 9 min 45 s (15 cycles), 68 °C for 11 min (15 cycles) and 68 °C for 13 min (15 cycles). A final extension step was performed at 68 °C for 6 min. PCR products were evaluated by agarose gel electrophoresis and stored at 4 °C.

{blacksquare} Nested PCR.
Based upon alignment of published HAV genome sequences, we selected conserved primer pairs (Table 1) to generate fragments representing the 5' UTR and the P1, P2 and P3 coding regions of the SLF88 genome. Fragments that overlapped these genome regions were then amplified using specific primer pairs (Table 1), designed using direct sequence information. PCR was performed in a 9600 Thermal Cycler using the following conditions: hot start at 94 °C for 90 s, denaturation at 94 °C for 35 s, annealing at 55 °C for 30 s and extension at 68 °C for 3 min. A total of 45 cycles was used with a final extension step at 68 °C for 6 s.

{blacksquare} Sequencing.
All fragments were sequenced in both directions using the primer-walking approach. Dye terminator reactions with rhodamine or drhodamine (PE Applied Biosystems) were electrophoresed using the ABI 377 or 373 automated sequencer (PE Applied Biosystems).

{blacksquare} Sequence data analysis.
Algorithms within the Wisconsin Package, version 10.1 [Genetics Computer Group (GCG)] were used for alignment of nucleotide sequences. Initial alignments were made using the GCG Pile-Up program; further adjustment to the alignments was performed manually using visual correction. Visual sequence comparison was performed with the Pretty program in GCG. Calculation of nucleotide and amino acid identities, calculation of genetic distances between sequences and construction of phylogenetic trees was performed by the computer software MEGA2 (Kumar et al., 1993 ). For the full-length genome sequence or all positions of codons for the P1 region, genetic distances were calculated by the Jukes–Cantor method (Jukes & Cantor, 1969 ). Genetic distances for synonymous and nonsynonymous substitutions of coding regions were calculated by the Nei–Gojobori method (Nei & Gojobori, 1986 ). Phylogenetic trees were constructed by the neighbour-joining method (Saitou & Nei, 1987 ). To confirm the reliability of the trees, bootstrap resampling tests were performed 1000 times (Felsenstein, 1985 ).


   Results
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Abstract
Introduction
Methods
Results
Discussion
References
 
Epidemiological background and serological testing
In the spring and summer of 1988, two young female missionaries who were associated with a rural hospital in Sierra Leone, Africa, both developed fulminant hepatitis A. The first patient, who had not received immune globulin since May 1987, developed clinical symptoms of hepatitis (12–15 May 1988). Within a week, she became delirious and incontinent and was medically evacuated to The Netherlands, but died 24 h after arrival. The necropsied liver from this case was the source of virus used for amplification and sequencing in this study. All contacts of this patient were given gamma globulin on 26 May 1988, with the exception of the second patient who was 7·5 months pregnant and declined prophylactic treatment. On 6 July 1988, the second patient delivered on the same mattress as that used by the first patient. The second patient developed clinical symptoms of hepatitis on 31 July 1988, which progressed to fulminant disease and resulted in coma within a week. She was flown to The Netherlands, where hepatitis A was confirmed by serological testing and where, after intensive treatment, she survived.

Since the two cases had no direct contact and the time between the first and the second case exceeds the usual incubation period, other sources linking the infection between these two patients were investigated. The most likely source of infection for the second patient was residual virus in the sponge-like mattress upon which the first patient had been incontinent. The surface of the mattress had been cleaned, but reports indicate that urine and diarrhoea soaked through the absorbent sponge centre to the box frame supporting the mattress.

Full-length genome amplification
Earlier studies on the virus responsible for these cases revealed that it was a distinct genotype, designated genotype VII, based upon a 170 bp sequence within the VP1/P2A junction region (Robertson et al., 1992 ). In this investigation, we amplified a full-length HAV amplicon, about 7·5 kb, using the liver-derived RNA (Fig. 1a). This full-length product was then used as a template to amplify the 5' UTR and the P1, P2 and P3 genome regions (Fig. 1b) using the conserved primer pairs identified in Table 1. Specific primer pairs (Table 1) designed on the basis of direct sequence information from these regions were then used to generate fragments that spanned the junctions (Fig. 1b).



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Fig. 1. Full-length genome amplification and a schematic illustration of the PCR amplicons used for sequencing. (a) Agarose gel analysis of the full-length genome product. Lane M, marker DNA with the length (kb) indicated to the left; lane A, full-length cDNA product from liver-derived RNA. (b) Schematic representation of the HAV genome and the amplicons used for sequence determination. The 5' UTR and the P1, P2 and P3 genome regions are depicted on top, while the individual amplicons that represent these regions are shown below, along with the amplicons that span the junctions of these regions. The numbers above each line indicate the terminal nucleotide of the respective primers used to amplify these products.

 
Nucleotide comparison and phylogenetic relationship of genotype VII sequence to other HAV sequences
The genome sequence that we determined included 7414 bases, nt 32–7472 (based upon HM-175 alignment). There are complete sequences available for representatives of human HAV genotypes IA and IB and simian HAV genotype V (AGM27). As shown in Table 2, the SLF88 sequence is about 86% identical to the wild-type representatives of human HAV genotypes IA (X75215, GBM) and IB (M14707, HM-175) and 82% identical to the wild-type simian HAV genotype V (D00924, AGM27). These values do not differ when the three coding regions are considered independently; however, 5' UTRs are more conserved, with about 95% identity for the human HAV genotype IA and IB sequences and 88% identity with the simian HAV genotype V sequence.


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Table 2. Nucleotide and amino acid identity of SLF88 relative to representative strains of human HAV genotypes IA and IB and simian HAV genotype V

 
For genotypes IIIA and IV, capsid sequences only are available. Fig. 2 illustrates the phylogenetic analyses of the capsid region (nt 735–3107) using all nucleotide positions (Fig. 2a), synonymous positions only (Fig. 2b) or nonsynonymous positions only (Fig. 2c). Synonymous and nonsynonymous positions were evaluated independently to determine the differential evolution reflecting mutations resulting from the absence and presence of selective pressure. The trees representing all positions and only nonsynonymous positions resulted in the SLF88 branch falling adjacent to human HAV genotypes IA and IB, with bootstrap support of 96–99% for this clustering. In contrast, analysis of only synonymous positions grouped SLF88 with the simian AGM27 strain; however, the bootstrap value supporting this relationship was only 41%.



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Fig. 2. Phylogenetic trees using all available P1 genome region sequences. (a) All nucleotide positions were used for this analysis. (b) Phylogenetic tree using only synonymous nucleotide positions for analysis. (c) Phylogenetic tree using only nonsynonymous nucleotide positions for analysis. Horizontal bars indicate genetic distance. Roman numerals (I, III, IV, V, and VII) above each branch indicate genotypes. Bootstrap values greater than 70% are indicated at the appropriate nodes. Accession numbers for the sequences included are as follows: HAS15 (X15463 and X15464); FG (X83302); GBM (X75215); HM-175 (M14707); MBB (M20273); SLF88; and AGM27 (D00924); CR326 (M10033); PA21 (M34084); GA76 (M66695); CY-145 (M59286).

 
Amino acid comparison within the three genome regions
We aligned available amino acid sequences for the P1 capsid region and identified amino acids changes relative to HM-175. These results are illustrated in Fig. 3(a). The SLF88 sequence contains about the same number of amino acid changes as members of genotype IA and all of the amino acid changes relative to HM-175 are conservative (lysine/arginine and threonine/serine). The numerical values for the amino acid identity of SLF88 to the P1 capsid region within AGM27, GBM and HM-175 (all wild-type strains) are shown in Table 2 and reflect the graphical data shown in Fig. 3(a).



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Fig. 3. Amino acid differences between individual HAV strains and the HM-175 human prototype sequence in the (a) P1, (b) P2 and (c) P3 genome regions. The horizontal line indicates identity with the HM-175 sequence, while the vertical lines indicate positions of changed amino acids. HM-175 represents genotype IB, GBM represents genotype IA, PA21 represents genotype IIIA and AGM27 and CY-145 are simian HAV strains representing genotypes V and IV, respectively. Immunodominant amino acids (arrows) associated with the major neutralization epitope of the virus capsid are indicated.

 
Amino acid comparison within the P2 genome region for the sequences that are available suggest that there are more overall amino acid changes in this region (Fig. 3b). As is the case in the P1 genome region, there is no distinct pattern that separates genotypes IA and IB or the SLF88 genotype VII, and the majority of the amino acid differences in SLF88 relative to HM-175 are homologous (valine/isoleucine, lysine/arginine and glutamic acid/aspartic acid). The numerical values of the SLF88 amino acid identities relative to the two human wild-type sequences GBM and HM-175 are between 96 and 97%, while comparison to AGM27 reveals 91·8% identity (Table 2). These values do not differ greatly from the analogous comparison in the capsid region discussed above.

In contrast, the P3 genome region contains numerous amino acid changes when the SLF88 sequence is compared to HM-175 (Fig. 3c) and the majority of these changes occur within the 3D polymerase. This pattern was distinct from that seen with genotype IA and IB sequences in which limited amino acid differences were detected. There appear to be more differences overall and we identified a total of 34 changes compared to HM-175. A more detailed inspection of these changes revealed that 45% were homologous amino acid changes when compared to HM-175. Despite the fact that the overall nucleotide identities within the P3 genome region did not differ compared to the values for the P1 and P2 genome regions, the amino acid identities in the P3D region were consistently lower, 90–93% compared with 96% for the remainder of the coding regions (Table 2). Many of the changes detected within the amino acid sequence of the P3D polymerase region were the result of nonsynonymous mutations; this phenomenon was not seen in the remainder of the coding region (data not shown).


   Discussion
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Abstract
Introduction
Methods
Results
Discussion
References
 
Hepatitis A is normally an acute disease with no chronic sequelae and a low fatality rate. Fulminant hepatitis A is rare and is calculated to occur in 0·14–0·35% of hospitalized cases (McNeil et al., 1984 ; Papaevangelou et al., 1984 ); there are no published studies that characterize the aetiology of fulminant hepatitis A infection. We determined the complete genome sequence of the wild-type SLF88 strain because epidemiological investigation of these cases provided evidence of indirect transmission of the virus between patient one and patient two, and the fulminant nature of both infections suggested that a genetic feature of the virus might be responsible. In addition, the SLF88 strain is the only member of genotype VII identified. We found that the capsid amino acid sequence was conserved when compared to genotype I viruses, but an increasing number of amino acid changes could be detected in the P2 and P3 nonstructural regions compared to genotype I viruses. Additional sequences are needed from HAV that circulate in this part of Africa to derive conclusions regarding the contribution of this genotype to fulminant disease.

One of the characteristics of HAV that facilitated transmission in this investigation is the extreme resistance of HAV to environmental conditions, which has been shown in the laboratory to remain infectious in the dried state for 1–2 months (McCaustland et al., 1982 ; Abad et al., 1994 ). In retrospect, the mattress used by the first patient was probably the source of virus for infection of the second patient, who, in the process of giving birth, transferred the virus from the mattress to her hands and mouth and, thus, developed the disease. These cases illustrate also the importance and efficacy of gamma globulin to prevent disease at a time when vaccine is not available. The first patient had not received immune globulin since May 1987 and, therefore, was susceptible to infection. The second patient was the only contact who did not receive immune globulin after the death of the first patient.

Genetic characterization of established HAV genotypes has focused traditionally on the VP1 amino terminus or the VP1/P2A junction region, where a reference database of sequences is available. However, there are genotypes (II, IIIA, IIIB, IV, V and VII) that have quite distinct genetic patterns compared to human HAV genotypes IA and IB in the regions that have been evaluated. The complete genome information we obtained in this study indicates that the nucleotide identity between genotype VII (SLF88) and other human HAV genotypes and the simian HAV sequences are about 86 and 82%. These numerical values are consistent with the genotype assignment based upon the VP1/P2A junction region and phylogenetic analysis confirms that SLF88 is a distinct HAV genotype.

Phylogenetic analysis of all sequences used in this study for the P1 region provided an interesting observation. The trees based upon all positions or only synonymous positions contain (i) a branch with human HAV strains (genotypes I and VII) and simian HAV strain AGM27 and (ii) a branch that includes human HAV genotype III and simian HAV strain CY-145. These two simian strains on separate branches may suggest separate evolutionary pathways that do not reflect host-dependent co-evolution. In contrast, the tree based upon nonsynonymous positions contains a branch that includes genotypes found in human hepatitis A infections, as distinct from simian HAV strains. This is consistent with host-specific evolution among human HAV strains and may reflect changes for specific virus–host interactions, such as that with the immunodominant neutralization epitope (Ping et al., 1988 ; Nainan et al., 1992 ). Further elucidation of this apparent discrepancy may be clarified by additional full-length genome sequences from other HAV genotypes.

An inspection of the translated amino acids in the three genome regions revealed differences in the types of amino acid changes within these regions. Within the P1 and P2 regions (1422 total aa), 16 of 21 (76%) amino acid changes were homologous amino acid substitutions. In contrast, within the P3 region (805 aa), there were 34 amino acid changes, of which 80% (n=27) were in the 3D polymerase region and which contained only 13 homologous substitutions. An evaluation of synonymous and nonsynonymous nucleotide changes throughout the coding region revealed that within the P3D region, nonsynonymous changes predominated, while synonymous changes were suppressed. A suppression of synonymous nucleotide changes in the analogous genome region of HCV RNA has been suggested to result from structural constraints (Smith & Simmonds, 1997 ), but the concomitant increase in nonsynonymous nucleotide changes that we have observed is not consistent with this explanation. It is possible that the mutations that we have found would alter polymerase efficiency, resulting in increased replication and more aggressive disease. If this is true, chimeras containing the polymerase region from SLF88 with characterized HM-175 sequences could be used to address this question.

Most of the viruses used for genotype identification (Robertson et al., 1992 ) were derived from regions of the world where hepatitis A is not endemic. This is a reflection of being able to identify the disease and, therefore, to obtain the biological samples needed for virus detection. Within hyper-endemic regions, such as the Amazon basin in South America, most of Africa, the Middle East and Central Asia and the Indian subcontinent, the majority of infections occur during childhood (Hadler, 1991 ). In these areas, distinct outbreaks occur rarely and clinical disease related to HAV infection is uncommon, as children generally experience asymptomatic infection. Genotype VII SLF88 is the only wild-type virus from this part of Africa for which we have a complete genome sequence. Further characterization of the genetic characteristics of HAV from different regions of Africa is needed to help us understand the molecular epidemiology of HAV in this hyper-endemic area.


   Acknowledgments
 
We would like to acknowledge the following individuals for their contributions to this study. From Sierra Leone, Africa: Amara Luckay, Stephen Gborie, Kandeh Kargbo and Drs George Komba-Kono, T. K. Kargbo, R. Baker and D. Metzeger who assisted with the epidemiological field investigation. Harbour Hospital, Rotterdam, Holland: Drs P. C. Stuiver, J. K. Doorduyn, J. L. J. Gaillard, D. J. Bac, P. R. Oosting, A. M. Dumas and J. L. M. Goud who provided clinical information for both patients and the autopsy of patient one. Central Public Health Laboratory, London, UK: David Brown who confirmed the absence of markers for viral haemorrhagic fever and determined the serological evidence for acute HAV infection. Centers for Disease Control and Prevention, Division of Viral and Rickettsial Diseases, Atlanta, GA, USA: Susan Fischer-Hock and Gilda Perez-Oronoz in the Special Pathogens Branch who tested for viral haemorrhagic fever viruses; the Hepatitis Reference Laboratory, Howard Fields and Kris Krawczynski, Hepatitis Branch, who confirmed the serology for hepatitis viruses and identified the presence of hepatitis A virus antigen in the necropsied liver tissue of patient one by IFA, respectively, and Harold S. Margolis and Miriam J. Alter for consultation during the course of the field investigation.


   Footnotes
 
The sequence of the SLF88 genotype VII strain of HAV has been deposited into GenBank under the accession no. AY032861.

b Present address: Office of the Director, Division of AIDS, STD and TB Laboratory Research, National Center for Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, GA, USA.

c Present address: Global AIDS Program, National Center for HIV, STD and TB Prevention, Atlanta, GA, USA.


   References
Top
Abstract
Introduction
Methods
Results
Discussion
References
 
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Received 22 June 2001; accepted 20 September 2001.