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
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Abstract |
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Introduction |
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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 7·07·5%. 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.
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Methods |
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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 RNAprimer 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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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.
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).
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 JukesCantor method (Jukes & Cantor, 1969
). Genetic distances for synonymous and nonsynonymous substitutions of coding regions were calculated by the NeiGojobori 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
).
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Results |
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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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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, 9093% 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).
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Discussion |
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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 12 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 virushost 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.
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Acknowledgments |
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Footnotes |
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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.
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Received 22 June 2001;
accepted 20 September 2001.