Intrinsic Signals for the Assembly of Hepatitis A Virus Particles
ROLE OF STRUCTURAL PROTEINS VP4 AND 2A*

Christian ProbstDagger , Monika Jecht§, and Verena Gauss-Müller

From the Institute of Medical Microbiology and Hygiene, Medical University of Lübeck, 23538 Lübeck, Germany

    ABSTRACT
Top
Abstract
Introduction
References

Capsid assembly is the final event of virus replication, and its understanding is pivotal for the design of empty capsid-based recombinant vaccines and drug delivery systems. Although the capsid structure of several members of the picornavirus family has been elucidated, little is known about the structural elements governing the assembly process that is tightly associated with proteolytic processing of the viral polyprotein. Among the picornaviruses, hepatitis A virus (HAV) is unique in that it contains VP1-2A as a structural component and the small structural protein VP4, which argues for an assembly pathway different from that proposed for other picornaviruses. Using a recombinant system we show here that proteolytic processing of the HAV capsid proteins' precursor P1-2A is independent of the terminal domains 2A and VP4 of the substrate. However, both terminal domains play distinct roles in the assembly of viral particles. 2A as part of P1-2A is a primary signal for the assembly of pentameric structures which only further aggregate to empty viral capsids when VP4 is present as the N terminus of the precursor. Particle formation in the hepatovirus genus is thus regulated by two intrinsic signals that are distinct from those described for other picornaviruses.

    INTRODUCTION
Top
Abstract
Introduction
References

Based on crystallographic data, the capsid structure of several members of the picornavirus family has been determined in detail (see Ref. 1 for review). The mature icosahedral capsid is composed of 12 pentamers, which comprise five copies each of the four viral structural proteins, VP1, VP2, VP3, and VP4. The particle's 5-fold axis of symmetry comprises parts of 5 molecules of the major viral capsid protein VP1 and is stabilized by an extensive network formed by the N termini of proteins VP1, VP3, and VP0, the precursor polypeptide of VP2 and VP4. At the 3-fold axis of the mature virion, the N termini of three VP2 molecules interact, and at the 2-fold axis VP2 and VP3 of neighboring protomers come in contact (2). The smallest picornaviral capsid protein VP4, which is liberated during the final step of capsid maturation, is found on the inside of the capsid shell facing the viral RNA genome. The myristoylated N terminus of VP4 is located below the 5-fold vertices, whereas its C terminus is found at the 3-fold axis and thus near the N terminus of VP2. Based primarily on structural analyses of mature viral particles and on disassembly studies, it has been concluded that protomers that consist of one molecule of VP1, VP3, and VP0 and sediment at 5 S presumably assemble into 14 S pentamers (1, 3). For poliovirus, the prototype of the enteroviruses, it was convincingly shown by genetic studies that myristoylation at the N terminus of VP4 and/or its precursors is a prerequisite for assembly into 14 S pentameric and higher ordered structures (4, 5). In addition, VP4 seems to be crucial for the release of viral RNA from the mature virion following adsorption to target cells (6). Assembly of the picornaviral capsid depends on the proteolytic liberation of the individual structural proteins from the precursor P1 or P1-2A catalyzed by proteinase 3Cpro, which is encoded within the P3 domain of the viral polyprotein. The genetic map of hepatitis A virus (HAV)1 with the position of the viral structural proteins in P1-2A and the viral proteinase 3Cpro in P3 is illustrated in Fig. 1 and at the top of Fig. 5.

For the mature particles of HAV no crystallographic data are available. In vivo studies on morphogenesis suggested that assembly of HAV particles might be different from that of other picornaviruses (7-9, 11). In particular, maturation cleavage of HAV VP0, which yields VP2 and VP4 and generally converts noninfectious into stable infectious virions, seems to be protracted (9). In contrast to most picornaviruses, HAV structural protein VP4 is not myristoylated and only about one-third in length (10). Possibly due to its small size, VP4 has so far not been detected in HAV particles. If present, it is rather unlikely that a protein as small as HAV VP4 fulfills the same structural role as the homologous but considerably larger polypeptide of other picornaviruses that extends from the 3-fold to the 5-fold axes of symmetry in the virion. Furthermore, HAV empty capsids and early assembly intermediates contain VP1-2A instead of VP1 (7, 9, 11, 13, 20). Whereas protein 2A of entero- and rhinoviruses functions as proteinase, the corresponding HAV protein is proteolytically inactive and forms part of the putative structural proteins' precursor P1-2A (1, 14). Based on these observations, HAV 2A can be considered a structural component and might thus be involved in morphogenesis. The HAV structural proteins (VP0, VP3, VP1, or VP1-2A) are released by proteolytic liberation from the precursor polypeptide P1-2A, which represents the N-terminal third of the viral polyprotein (for genomic map, see top of Fig. 5).

In this study, we examined the role of the unique terminal domains (VP4 and 2A) of the HAV structural proteins' precursor P1-2A using a genetic in vivo approach. HAV P1-2A and its derivatives truncated at the N or C terminus were coexpressed with P3 as the source of the viral proteinase. Irrespective of the presence of the terminal protein domains, liberation of the structural proteins was equally efficient. However, the assembly of HAV particles was dependent on both terminal domains 2A and VP4 and is thus different from other picornaviruses. A model for the assembly of HAV particles is proposed.

    EXPERIMENTAL PROCEDURES

cDNA Constructs-- Construction of plasmids pEXT7-HM/HAS-P1-2A(E/S) and pEXT7-HM/HAS-P1[273] was described previously (15). For simplicity the proteins encoded by these constructs are here called P1-2A and P1, respectively. To ensure efficient processing of the VP1/2A site, the E/S cleavage site of HAV strain HAS-15 at VP1 amino acid position 273 was used (15). To construct a plasmid encoding the VP4-deleted precursor polypeptide, pT7-HAV1 (GenBankTM accession number M16632, Ref. 16) was used as template for polymerase chain reaction amplification with primers 5'-TTATGGCCATGGACATTGAGGAAGAGCAAATGATTCAATCAG-3' (sense 5'-VP2) and 5'-TCTGGTCACCAGGAACCATAGCACAGATCAATCCCCCC-3' (antisense 3'-VP2). After restriction with MscI and BstEII (underlined), the 0.4-kilobase pair fragment representing nucleotides 799 to 1189 of the attenuated HAV strain HM175 was inserted into pEXT7-HM/HAS-P1-2A(E/S) cut with the same enzymes. The resulting construct (pEXT7-HM/HAS-VP2-2A(E/S)) encodes the VP4-deleted polypeptide VP2-2A comprising amino acids 24 to 836 of the HAV polyprotein. Fig. 1 shows the genetic elements of all three P1-2A constructs. pGEM2-HM-P3, which has been described elsewhere, was used as the source of proteinase 3Cpro (12). The nucleotide sequences of the polymerase chain reaction-amplified regions were verified by DNA sequencing.


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Fig. 1.   HAV P1-2A cDNA constructs used for coexpression with pGEM2-HM-P3 in the vaccinia virus system. The HAV coding sequence is preceded by the T7 promoter, the internal ribosomal entry site of encephalomyocarditis virus (EMCV IRES), and four additional amino acid residues as marked. E/S refers to the amino acid sequence at the VP1/2A cleavage site described before (15). Poly(A) is derived from the SV40 T antigen; Tphi , T7 terminator.

Expression and Analysis of Viral Proteins and Particles-- For expression 3 × 105 COS7 cells grown overnight to approximately 70% confluency in wells of 35 mm diameter were transfected with a total of 1 µg of purified cDNA and 9 µl of LipofectAMINETM according to the instructions of the manufacturer (Life Technologies, Inc.). 3 h after transfection, infection with the recombinant vaccinia virus vTF7-3 (1 multiplicity of infection) followed for 30 min at 37 °C (17). After replacing the medium by 2 ml of Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, incubation was continued for 18 h. Then the cells were scraped off the plate in 300 µl of phosphate-buffered saline containing 0.05% (v/v) Tween 20. 70 µl of the crude cellular extract were separated on a discontinuous 12% SDS gel and blotted onto nitrocellulose. Colorimetric detection of recombinant proteins was performed with polyclonal anti-VP0 (18), anti-VP4 (19), anti-VP3/VP1 (18), anti-VP1 (18), anti-2A (15), anti-3C sera (14), and anti-immunoglobulins conjugated to alkaline phosphatase. Since the anti-2A serum was raised against a purified recombinant polypeptide spanning amino acid residues 255-345 of VP1-2A of the attenuated strain HM175 and thus including the 19 C-terminal amino acids of VP1[273] (15, 20), minor immune reactivity with VP1-containing proteins was observed (see lane 13 in Fig. 2). For particle detection, the crude extracts were clarified by centrifugation. Then the supernatants were diluted 1:10 in phosphate-buffered saline containing 0.05% (v/v) Tween 20 and analyzed by an ELISA using the neutralizing monoclonal antibody K2-4F2 as capture and detection antibody (21-23). Each transfection assay was performed twice and analyzed in duplicate. Rate zonal centrifugation was performed as described before (7, 11). Briefly, after lysis of 1 × 107 cells in 1 ml of NPT (100 mM NaCl, 0.5% (v/v) Nonidet P-40, 10 mM Tris, pH 7.3) the extracts were clarified by centrifugation at 13,000 rpm for 5 min and adjusted to 1% (w/v) SDS. Ultracentrifugation was performed in a SW41 rotor (Beckman Instruments) at 35 krpm, 4 °C for 3 h using a linear 5-30% (w/w) sucrose gradient in NT (100 mM NaCl, 10 mM Tris-HCl, pH 7.3). The gradient was fractionated from the bottom of the tube. 50 µl of each fraction (1 ml total) were analyzed by an ELISA using the neutralizing monoclonal antibody. For immunodot analysis, 450 µl of each fraction were made 0.4% in SDS, boiled for 10 min, and dotted on nitrocellulose. The subsequent immune reaction with anti-VP1 and anti-2A was performed as described above and expressed in relative units. The sucrose concentration of each fraction was measured refractometrically.

    RESULTS AND DISCUSSION

Viral Proteinase 3Cpro-mediated Cleavage of Hepatitis A Virus Polypeptide P1-2A and Its VP4- or 2A-deleted Forms (VP2-2A and P1)-- Polypeptide P1-2A is the putative functional precursor of the HAV structural proteins that is cleaved by the viral proteinase 3Cpro contained in domain P3 of the viral polyprotein. To study the role of the N- and C-terminal regions of P1-2A (VP4 and 2A, respectively) in both proteolytic cleavage and particle formation, the HAV precursor polypeptide P1-2A and VP4- and 2A-deleted forms (VP2-2A and P1, Fig. 1) were transiently expressed in the presence of the P3 domain using vaccinia virus vTF7-3 as helper virus. By cotransfection of two T7-promoted cDNA constructs, we could exclude viral genome replication and focus our studies on protein synthesis and processing and particle assembly. Proteolytic cleavage efficiency was analyzed by immunoblot using antisera directed against purified HAV proteins. For the individual precursor proteins (lysates B-D), immune reaction with anti-VP1 shows that expression of P1-2A (lysate B), VP2-2A (lysate C), and P1 (lysate D) was equally efficient and generated the respective unprocessed structural precursor molecules (Fig. 2, lanes 5-7). The lack of immunoreaction of the P3 extract (lysate A, lane 4) demonstrates the specificity of the anti-VP1 serum. The cellular lysates obtained after coexpression of P1-2A (lysate E), VP2-2A (lysate F), or P1 (lysate G) with P3 were analyzed for proteins reactive with anti-VP1 (Fig. 2, lanes 8-10) and anti-2A (Fig. 2, lanes 11-13), anti-VP0 (Fig. 3, lanes 1-3), anti-VP4 (Fig. 3, lanes 4-6), anti-VP3/VP1 (Fig. 3, lanes 7-9). As additional control for efficient coexpression of P3, lysates E, F, and G were analyzed for the presence of 3C-containing proteins, which are derived from autoproteolysis of P3 (see Ref. 12, Fig. 2, lanes 1-3). Almost equal intensities of the bands 3C, 3BC, and 3ABC clearly demonstrate equal amounts of coexpressed and autoproteolytically active P3. The immunoblot with anti-VP1 and anti-2A of lysates E to G showed the expected processing products. VP1, VP1-2A, and VP3-VP1-2A were found as products of P1-2A (lysate E) and VP2-2A (lysate F), whereas the 2A-containing polypeptides were lacking among the products derived from P1 (lysate G), clearly demonstrating that correct processing occurred in this experimental system. The lysates probed with anti-VP0 and anti-VP4 showed processing products VP0-VP3, VP2-VP3, VP0, and VP2, which were identified by their electrophoretic mobility and by comigration with proteins of different immunoreactivity (Fig. 3). A nearly identical staining pattern was obtained by immunoreaction with anti-VP3/VP1 (Fig. 3). The specificity of the reaction with anti-VP4 was clearly demonstrated by the lack of any reactivity of lysate F (Fig. 3, lane 5). Based on these observations we conclude that the additional protein bands in Fig. 3 are aberrant translation or processing products and do not represent unspecific binding of the antisera. Taken together, it is obvious that P3-mediated cleavage of the HAV structural proteins' precursor P1-2A is efficient and is not affected by deletions of VP4 or 2A. Specific processing was not observed in the absence of P3 (Fig. 2, lanes 5-7) nor was cleavage of VP0 into VP4 and VP2 detected (Fig. 3, lanes 1-3).


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Fig. 2.   Proteolytic processing and HAV antigen production as determined by ELISA and immunoblot analysis after coexpression of HAV P3 with P1-2A-derived polypeptides. As shown in the center of the figure by dots, recombinant P1-2A proteins were either expressed alone (lysates B-D) or together with P3 encoding the viral proteinase 3Cpro (lysates E-G). In the immunoblot with anti-VP1, anti-2A, and anti-3C, the viral proteins were identified on the basis of their electrophoretic mobility and their combined immunoreactivity. Immunoreactive products are marked on both sides, the position of molecular mass standards is shown on the left. Upper panel, particle formation was determined by ELISA using the neutralizing monoclonal antibody K2-4F2. For the ELISA, each transfection was performed twice and analyzed in duplicate. As negative controls, the precursor proteins P1-2A, VP2-2A, P1, or P3 were expressed separately. The specific antigenicity is expressed as the mean ELISA signal from the different sets of coexpressions (lysates E-G) normalized by the mean value of the negative controls (lysates A-D). Error bars indicate the standard deviation.


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Fig. 3.   Immunoblot analysis after coexpression of HAV P3 with P1-2A-derived polypeptides. As described for Fig. 2 and marked by the dots on top of the figure, HAV proteins were expressed and analyzed with anti-VP0, anti-VP4, and anti-VP3/VP1.

Assembly of HAV Particles Derived from Expression of P1-2A and Truncated Precursors-- Aliquots of the same cell extracts used in the immunoblot experiments described above were further analyzed for HAV particle formation using an antibody specific only for particulate structures (upper panel of Fig. 2). The murine monoclonal antibody K2-4F2 used in the ELISA is directed against an immunodominant antigenic site that is essential for neutralization of viral infectivity. The conformation-dependent epitope involves amino acid residues of both VP3 and VP1 (21-23). Neither 2A nor VP4 seem to be essential constituents of the epitope since mature infectious particles lacking 2A are efficiently neutralized by the monoclonal antibody K2-4F2 (23), and coexpression of the VP4-deleted precursor (VP2-2A) with P3 led to the formation of structures recognized by K2-4F2 (see below). In combination with sedimentation analyses, this monoclonal antibody is thus appropriate to monitor viral assembly steps subsequent to the initial association of uncleaved precursor polypeptides (see also Fig. 5 for the reactivity of K2-4F2). No K2-4F2-reactive antigen was produced when the precursor polypeptides were expressed in the absence of the proteinase implying that the uncleaved precursors do not form the neutralizing epitope (Fig. 2, lysates B, C, and D). The absence of K2-4F2 reactivity with uncleaved precursor polypeptides demonstrates the specificity of the monoclonal antibody in distinguishing between processed and assembled HAV structural proteins on one side and their precursor on the other. The highest antigenic reaction was detected after coexpression of P1-2A with P3 (Fig. 2, lysate E). Although processing of the precursor polypeptides did not depend on the presence of either VP4 or 2A (see Figs. 2 and 3), particle formation decreased to near background level when 2A was deleted (Fig. 2, lysate G) and to approximately one-fifth when VP4 was deleted from the precursor (lysate F). From these results we conclude that VP4 and 2A play important roles in the assembly of HAV particles.

To characterize the sedimentation properties of the viral antigen, rate zonal centrifugation on a 5 to 30% (w/w) sucrose gradient was performed with extracts generated by coexpression of P1-2A, VP2-2A, or P1 with P3 (Fig. 4). The major antigenic product detected in the P1-2A + P3 lysate sedimented with approximately 70 S and thus has the form of empty capsids. Only minor quantities of antigenicity were identified in the 12% sucrose fraction (Fig. 4A). In contrast, the recombinant antigen produced by coexpression of VP2-2A + P3 showed a sedimentation profile characteristic for 14 S pentameric structures with virtually no empty capsids (Fig. 4B). No K2-4F2-reactive particles were detected in the P1 + P3 or control extract (Fig. 4, C and D). These data indicate that no aggregation occurs in the absence of 2A and that assembly of HAV pentameric structures to empty capsids is impaired in the absence of VP4. The presence of only small amounts of 14S pentamers in the P1-2A + P3 extracts suggests that aggregation of pentamers to empty procapsids is highly efficient (Fig. 4A). As expected, RNA-containing particles sedimenting faster than procapsids were not detected in this system, probably due to the lack of putative packaging signals exposed on the viral RNA.


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Fig. 4.   Characterization of assembly intermediates produced by recombinant coexpression of HAV P1-2A derivatives with P3 by rate zonal centrifugation and immunodot blot. COS7 cells were transinfected with the cDNAs as indicated (A-C). The control extract was prepared from vTF7-3 infected cells (D). After rate zonal centrifugation, fractions were analyzed by ELISA using the neutralizing monoclonal antibody K2-4F2 (indicated by full squares). For the immunodot blot, SDS was added to an aliquot of each gradient fraction (0.4% final concentration). After boiling for 10 min, the samples were dotted onto nitrocellulose. The subsequent immune reaction was performed as described in Fig. 2. The anti-VP1 immune reaction was quantified and expressed in relative units (indicated by open squares). The sucrose concentration of the fractions are indicated on top of the figure. Coexpression of P1-2A with P3 resulted in the formation of empty capsids (A), whereas the VP4-deleted construct (VP2-2A + P3) led to an accumulation of pentameric structures (B). The deletion of 2A (P1 + P3) drastically reduced the formation of K2-4F2-reactive antigens (C).

To determine the VP1- and 2A-specific antigenicity of HAV particles, the fractions of the sucrose gradients were analyzed for HAV structural proteins by a denaturing dot blot assay. Both VP1- and 2A-reactive proteins were found in the fractions containing empty capsids or pentamers (Fig. 4, A and B, respectively). In addition to the K2-4F2-reactive particles sedimenting at about 70 S, faster sedimenting particles were reactive with anti-VP1 and anti-2A (Fig. 4A). It is possible that the neutralization epitope of faster sedimenting particles in Fig. 4A is masked by either aggregation or altered conformation. For HAV particles, a wide range of sedimentation coefficients have been reported (7, 8), and it is not clear whether their formation depends on specific experimental parameters. Probably due to their heterogeneity, HAV particles could not be crystallized yet. No structural proteins were detected in fractions of gradient C shown in Fig. 4, confirming our previous observation that assembly of 14 S pentamers and subsequently empty capsids is abrogated when 2A is deleted from the polyprotein. From the experimental data presented here we can conclude that both terminal domains of HAV precursor protein P1-2A serve distinct functions in the formation of HAV 14 S pentameric structures and empty particles.

Based on our results with deleted precursor polypeptides, we now modify and extend the previously reported assembly model for HAV particles (3, 7, 11) that is shown in Fig. 5A. Panels B and C of Fig. 5 depict impaired HAV assembly due to the lack of either VP4 or 2A. The C-terminal domain of P1-2A, protein 2A, is required for the initial assembly step leading to the formation of uncleaved 13 S and of 14 S pentameric structures upon proteolytic cleavage by 3Cpro. Although no structural data are available for HAV, this observation implies that 2A as part of VP1-2A might be located near the 5-fold vertices on the outside of the pentamer and the empty capsid. In contrast, assembly of the poliovirus particle depends on myristoylated VP4, which is found near the 5-fold axes on the inside of the capsid shell (2). Based on the known structure of other picornaviruses with the N terminus of VP2 located near the 3-fold axis and on the small size of HAV VP4, we assume that this structural protein is found on the periphery of the HAV pentamer. We propose that VP4 as part of VP0 is required for the subsequent association of 12 pentamers at the 3-fold axis. Our data provide first experimental evidence that the terminal domains of the HAV and poliovirus structural proteins' precursor might have exchanged their roles in the initial assembly step. Whereas myristoylated VP4 of poliovirus P1 is required to initiate aggregation of pentamers, the 2A domain of HAV P1-2A signals this first step. Furthermore, our experimental data indicate that two distinct signals separately regulate HAV capsid formation. In a first step, the assembly of HAV pentamers requires the presence of 2A, and in a second step, VP4 is necessary for the formation of empty capsids. In mature infectious virions, neither VP0 nor VP1-2A was observed in significant amounts, suggesting that removal of both terminal domains of the primary processing product are essential for maturation (7-11). The autocatalytic cleavage of VP0, which liberates VP4, is common to all picornaviruses (1, 3). However, maturation of the HAV capsid apparently requires an additional cleavage step that is protease-catalyzed and presumably occurs on the outer surface of the particle.


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Fig. 5.   Model for the hepatitis A virus assembly pathway and the role of the terminal domains VP4 and 2A of P1-2A (based on Refs. 3, 7, and 11). Panel A, after 3Cpro-mediated primary cleavage, five molecules of P1-2A assemble to form 13 S pentamers, which are mediated by 2A. Upon proteolytic cleavage of assembled P1-2A, 14 S pentamers arise. Presumably due to rearrangements, these particles expose the neutralization epitope and are thus recognizable by the monoclonal antibody K2-4F2. 12 pentamers can aggregate at their 3- and 2-fold axes and form empty capsids. In the presence of newly synthesized viral RNA, the nucleic acid can be packaged by 14 S pentamers (not shown). Panel B, in the presence of 2A, the N-terminally truncated precursor VP2-2A can assemble into 14 S pentamers. Further aggregation at the 3- and 2-fold axes is not possible in the absence of VP4. Panel C, due to the lack of the primary assembly signal 2A, the precursor P1 does not assemble into pentamers.

Recently, it was reported that up to 15% of HAV 2A can be deleted from the complete viral genome without significantly affecting the viability of the resulting virus, which had a small plaque phenotype (24). In the light of our results this observation can now be explained by inefficient particle formation due to the lack of the complete primary assembly signal. Our data presented here clearly show that the structural protein precursor P1-2A in combination with P3 is sufficient for the recombinant expression and assembly of empty capsids exposing epitopes essential for neutralization. These recombinant, noninfectious particles might prove useful for the design of diagnostic tools and in addition for vaccine products. Possibly, our data and the proposed model of assembly also apply to particle production of mengovirus and foot and mouth disease virus both of which contain 2A as a protein of unknown function and have been considered as vectors for gene transfer.

    ACKNOWLEDGEMENTS

We thank Dieter Reinhardt for fruitful comments and suggestions on the manuscript. We are grateful to B. Moss for vaccinia virus vTF7-3 and Dr. H. Andres (Hoffmann-La Roche Inc.) for the monoclonal antibody.

    FOOTNOTES

* The work was supported by Deutsche Forschungsgemeinschaft (DFG) SFB 367, Project B7.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger Recipient of a fellowship granted by the Studienstiftung des Deutschen Volkes.

§ Supported by a grant of the state of Schleswig-Holstein.

To whom correspondence should be addressed: Institute for Medical Molecular Biology, Medical University of Lübeck, Ratzeburger Allee 160, 23538 Lübeck, Germany. Tel.: 49-451-500-4085; Fax: 49-451-500-3637; E-mail: gaussmue{at}molbio.mu-luebeck.de.

    ABBREVIATIONS

The abbreviations used are: HAV, hepatitis A virus; ELISA, enzyme-linked immunosorbent assay.

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Abstract
Introduction
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