Trypsin sensitivity of several human rhinovirus serotypes in their low pH-induced conformation

Five serotypes of human rhinovirus (HRV) were examined for sensitivity to trypsin at physiological pH, HRV1A, HRV2, and HRV14 were found to be resistant whereas in serotypes HRV49 and HRV89 degradation of VP2 was observed. However, exposure to low pH followed by neutralization, a treatment which causes irreversible conformational changes in the capsid, led to rapid cleavage by trypsin of VP1 in HRV1A, HRV2, and HRV49 at defined sites followed by degradation of VP2. In the case of HRV2, the cleavage site in VP1 was determined by direct protein sequencing and was shown to occur between Arg260 and Thr261, close to the C-terminus. HRV49 behaves similarly to HRV2 as expected from extensive sequence similarity in this region, whereas VP1 in HRV1A is most probably cleaved at a site closer to the C-terminus than that in HRV2. Although HRV14 contains the same amino acid pair present in HRV2 and HRV49, it was not cleaved under these conditions. HRV89, which lacks a basic residue at the corresponding position, was also insensitive. Examination of the cleavage site on the three-dimensional structural map of native HRV2 reveals that it is most probably buried inside the capsid and thus not accessible. Structural rearrangements of the viral capsid are thus necessary to account for the cleavage observed after low pH treatment.     

Antigenic variation of foot-and-mouth disease virus of serotype C during propagation in the field is mainly restricted to only one structural protein (VP1)

The primary structure of VP3, VP2 and VP4 capsid protein genes has been determined for six epizootiologically-related foot-and-mouth disease virus (FMDV) isolates of serotype C1, two of which presented immunogenic differences as determined by a cross-protection assay. The results obtained have been compared with those previously reported for the corresponding VP1 genes Martinez et al. (1988) Gene 62, 75-84. High rates of fixation of mutations have been estimated for the four capsid protein genes that ranged from 3.9 X 10(-4) to 4.5 X 10(-3) substitutions per nucleotide per year, with the highest values corresponding to VP1. Despite this genetic heterogeneity most of the amino acid exchanges are within the VP1 protein. Of the fourteen amino acid substitutions one was located in VP2 and two in VP3. Five out of the eleven amino acid exchanges that affected VP1 were located within residues 138-149, part of a main immunogenic site in FMDV. These results show that in the course of a foot-and-mouth disease outbreak, immunologically relevant amino acid substitutions occur mainly in viral capsid protein VP1.     

Detection of unusual mutation within the VP1 region of different re-isolates of poliovirus Sabin vaccine

In the present study, a genomic analysis of full VP1 sequence region of 15 clinical re-isolates (14 healthy vaccinees and one bone marrow tumor patient) was conducted, aiming to the identification of mutations and to the assessment of their impact on virus fitness, providing also insights relevant with the natural evolution of Sabin strains. Clinical re-isolates were analyzed by RT-PCR, sequencing and computational analysis. Some re-isolates were characterized by an unusual mutational pattern in which non-synonymous mutations outnumbered the synonymous ones. Furthermore, the majority of amino-acid substitutions were located in the capsid exterior, specifically in N-Ags, near N-Ags and in the north rim of the canyon. Also mutations, which are well-known determinants of attenuation, were identified. The results of this study propose that some re-isolates are characterized by an evolutionary pattern in which non-synonymous mutations with a direct phenotypic impact on viral fitness are fixed in viral genomes, in spite of synonymous ones with no phenotypic impact on viral fitness. Results of the present retrospective characterization of Sabin clinical re-isolates, based on the full VP1 sequence, suggest that vaccine-derived viruses may make their way through narrow breaches and may evolve into transmissible pathogens even in adequately immunized populations. For this reason increased poliovirus laboratory surveillance should be permanent and full VP1 sequence analysis should be conducted even in isolates originating from healthy vaccinees.     

Genetic and phenotypic characterization of mutants in four essential genes that map to the left half of HSV-1 UL DNA

Several HSV-1 proteins including the major capsid protein (VP5), two minor capsid proteins (VP11-12 and VP18.8), the alkaline nuclease and glycoprotein gH have been reported to be encoded by the left-most one-third of HSV-1 UL DNA. In this paper, we present physical mapping data and phenotypic analysis of six ts mutants whose mutations lie within this region and which collectively represent four functional complementation groups (1-6, 1-7, 1-10, and 1-26). In this study, mutants in complementation group 1-10 were found to be defective in the synthesis of viral DNA, late viral polypeptides, and the formation of mature capsid-like structures--properties characteristic of other ts mutants defective in functions required for viral DNA synthesis. Two DNA-positive mutants in complementation group 1-7 fail to induce capsid formation and probably possess mutations in coding sequences for VP5. Mutants in two other complementation groups (1-6 and 1-26) synthesize significant levels of viral DNA, late polypeptides, and capsids. The functions of the gene products represented by these mutants remain to be determined.     

The trypsin-sensitive RVER domain in the capsid proteins of minute virus of mice is required for efficient cell binding and viral infection but not for proteolytic processing in vivo.

Analysis of a series of mutations in the trypsin-sensitive RVER region of the amino terminal domain in the capsid proteins (VP1 and VP2) of the autonomous parvovirus, minute virus of mice (MVM), demonstrates that this sequence is not essential for proteolytic processing of VP2 into VP3 in vivo, but specific amino acids within this domain are important for viral infection. Analysis of the most deficient of these mutants, VP(delta 2842-2863), a 7-aa deletion of aa 159-165 in VP1 and 17-23 in VP2, has identified at least two steps in MVM infection in which this domain is important. VP(delta 2842-2863) was 3-fold defective in binding to murine A9(2L) cells and, when an equivalent amount of virus was bound to cells, additionally 10-fold deficient compared to wild-type in initiating a productive infection. However, in those cells effectively infected, VP(delta 2843-2863) replicated similar to wild-type. These results suggest that these seven amino acids constitute a region important for both binding and a subsequent step prior to the start of DNA replication such as viral uptake or transport to the nucleus.     

Complete sequence of the RNA genome of human rhinovirus 16, a clinically useful common cold virus belonging to the ICAM-1 receptor group

We report here the complete nucleotide sequence and predicted polyprotein sequence of HeLa cell-adapted human rhinovirus 16 (HRV16). This virus is more suitable than human rhinovirus 14 (HRV14) for clinical studies, and its growth and physical properties are favorable for biochemical and crystallographic analysis. The complete message-sense RNA genome of HRV16 is composed of 7124 bases, not including the poly(A) tail. An open reading frame, extending from base 626 to 7084 predicts a polyprotein containing 2152 amino acid residues. Comparison with other rhinovirus sequences shows HRV16 is much more representative of human rhinoviruses than HRV14. No apparent relationship was found between receptor group and amino acid sequence in VP1, the capsid protein bearing the binding site for the intercullular adhesion molecule-1 (ICAM-1) in both HRV14 and HRV16.Genbank accession number: L24917.     

Functional conservation of the hydrophobic domain of polypeptide 3AB between human rhinovirus and poliovirus

In this study we exchanged portions of the poliovirus type 1 (PV1) hydrophobic domain within the membrane-associated polypeptide 3AB for the analogous sequences from human rhinovirus 14 (HRV14). The sequence exchanges were based upon a previous report in which the 22 amino acid hydrophobic region was subdivided into two domains, I and II, the latter of which was shown to be required for membrane association (J. Biol. Chem. 271 (1996), 26810). Using these divisions, the HRV14 sequences were cloned into the complete poliovirus type 1 cDNA sequence. RNAs transcribed from these cDNAs were transfected into HeLa cell monolayers and used in HeLa cell-free translation/replication assays. The data indicated that 3AB sequences from PV1 and HRV14 are interchangeable; however, the substitutions cause a range of significant RNA replication defects, and in some cases, protein processing defects. Following transfection of RNAs encoding the domain substitutions into HeLa cell monolayers, virus isolates were harvested, and the corresponding viral RNAs were sequenced. The sequence data revealed that for the carboxy-terminal domain substitutions (domain II), multiple nucleotide changes were identified in the first, second, and third positions of different codons. In addition, the data indicated that for one of the PV1/HRV14 chimeras to replicate, compensatory mutations within poliovirus protein 2B may be required.     

Lack of quantitative correlation between inhibition of replication of rhinoviruses by an antiviral drug and their stabilization

Summary R 61 837, a new antirhinovirus compound, was able to protect several susceptible rhinoviruses against inactivation by mild acidification or heat. This observation strengthens the hypothesis that the drug exerts antiviral activity by a direct interaction with the viral protein capsid to stabilize the particle. However, the minimal concentrations necessary to inhibit either acetate or citrate or heat inactivation were different for each of five tested serotypes and we therefore conclude that stabilization and inhibition of replication are not causally linked but parallel events, both independently resulting from the binding of the drug to the viral capsid.Studies using drug resistant mutants of HRV 51 and HRV 9 confirmed this lack of quantitative correlation. The mutants were also shown to be cross resistant to a panel of seven different reference antirhinoviral drugs including SDS, WIN 51711, chalcone, dichloroflavan and MDL 20,610. This indicates that all these compounds bind to the same site corresponding to the hydrophobic pocket within the viral protein VP 1 -barrel structure of HRV 14.     

Human rhinovirus capsid dynamics is controlled by canyon flexibility

Quantitative enzyme accessibility experiments using nano liquid chromatography electrospray mass spectrometry combined with limited proteolysis and isotope-labeling was used to examine the dynamic nature of the human rhinovirus (HRV) capsid in the presence of three antiviral compounds, a neutralizing Fab, and drug binding cavity mutations. Using these methods, it was found that the antivirals WIN 52084 and picovir (pleconaril) stabilized the capsid, while dansylaziridine caused destabilization. Site-directed mutations in the drug-binding cavity were found to stabilize the HRV14 capsid against proteolytic digestion in a manner similar to WIN 52084 and pleconaril. Antibodies that bind to the NIm-IA antigenic site and penetrate the canyon were also observed to protect the virion against proteolytic cleavage. These results demonstrate that quantifying the effects of antiviral ligands on protein "breathing" can be used to compare their mode of action and efficacy. In this case, it is apparent that hydrophobic antiviral agents, antibodies, or mutations in the canyon region block viral breathing. Therefore, these studies demonstrate that mobility in the canyon region is a major determinant in capsid breathing.     

Antigenic and genetic analyses of human rotavirus with dual subgroup specificity.

In our previous study (S. Urasawa, T. Urasawa, K. Taniguchi, F. Wakasugi, N. Kobayashi, S. Chiba, N. Sakurada, S. Morita, O. Morita, M. Tokieda, T. Kawamoto, K. Minekawa, and M. Oseto, J. Infect. Dis. 160:44-51, 1989) of antigenic characterization of about 300 human rotavirus (HRV) isolates collected at different localities in Japan, we found 4 HRV isolates having unique antigenic and genetic constructions. The four strains possessed both subgroup I and subgroup II antigens, serotype 3 antigen, and a long RNA electropherotype. The reactivity pattern of these four HRV isolates with three monoclonal antibodies (MAbs) directed to an outer capsid protein, VP4, and with one MAb directed to an inner capsid protein, VP2, was clearly different from those of usual subgroup II HRVs having serotype 1, serotype 3, or serotype 4 specificity and a long RNA pattern, whereas their reactivity pattern was similar to that of strain K8 (subgroup II, serotype 1), which possessed unique VP4 and VP2 proteins. RNA-RNA cross-hybridization analysis indicated that while the four isolates were genetically distinct from the two genetic groups of HRV reported previously, i.e., the Wa family (strains KU, S3, and YO) and the DS-1 family (strain S2), they were closely related to strain K8, a strain having unique antigenic and genetic properties (K. Taniguchi, K. Nishikawa, T. Urasawa, S. Urasawa, K. Midthun, A. Z. Kapikian, and M. Gorziglia, J. Virol. 63:4101-4106, 1989).     

Second site mutations in the N-terminus of the major capsid protein (VP5) overcome a block at the maturation cleavage site of the capsid scaffold proteins of herpes simplex virus type 1.

VP5, the major capsid protein of herpes simplex virus type 1 (HSV-1), interacts with the C-terminal residues of the scaffold molecules encoded by the overlapping UL26 and UL26.5 open reading frames. Scaffold molecules are cleaved by a UL26 encoded protease (VP24) as part of the normal capsid assembly process. In this study, residues of VP5 have been identified that alter its interaction with the C-terminal residues of the scaffold proteins. A previously isolated virus (KUL26-610/611) was used that encoded a lethal mutation in the UL26 and UL26.5 open reading frames and required a transformed cell line that expresses these proteins for virus growth. The scaffold maturation cleavage site between amino acids 610 and 611 was blocked by changing Ala-Ser to Glu-Phe, which generated a new EcoRI restriction site. Revertant viruses, that formed small plaques on nontransformed cells, were detected at a frequency of 1:3800. Nine revertants were isolated, and all of them retained the EcoRI site and therefore were due to mutations at a second site. The second site mutations were extragenic. Using marker-transfer techniques, the mutation in one of the revertants was mapped to the 5' region of the gene encoding VP5. DNA sequence analysis was performed for the N-terminal 571 codons encoding VP5 for all of the revertant viruses. Six of the nine revertants showed a single base pair change that caused an amino acid substitution between residues 30 and 78 of VP5. Three of these were identical and changed Ala to Val at residue 78. The data provide a partial map of residues of VP5 that alter its interaction with scaffold proteins blocked at their normal cleavage site. The yeast two-hybrid system was used as a measure of the interaction between mutant VP5 and scaffold molecules and varied from 11% to nearly 100%, relative to wild-type VP5. One revertant gave no detectable interaction by this assay. The amount of UL26 encoded protease (VP24) in B capsids for KUL26-610/611 and for revertants was 7% and 25%, respectively, relative to the amount in capsids for wild-type virus. The lack of retention of the viral protease in the mutant virus and a fourfold increase for the revertants suggest an additional essential function for VP24 in capsid maturation, and a role in DNA packaging is indicated.     

Molecular characterization of a variant rhinovirus from an outbreak associated with uncommonly high mortality.

BACKGROUND: Human rhinoviruses (HRVs) are the most frequent cause of acute upper respiratory tract infection, however, they are also known to replicate in the lower respiratory tract and associate with more severe respiratory illnesses. An outbreak of HRV occurred in a long-term facility in Santa Cruz, California with unusually high morbidity and mortality. OBJECTIVES: To identify viral characteristics associated with this unique outbreak, genetic relationships between these clinical isolates (SCRVs) and prototype strains of rhinovirus were investigated. STUDY DESIGN: Sequence homology and phylogenetic analyses of the SCRV VP4/VP2 region were performed in conjunction with all HRV prototypes. Due to the importance of the 5'noncoding region (NCR) and the structural genes to viral replication and host immune responses, respectively, we focused on a segment of the HRV genome which includes these regions. Molecular models of SCRV were also assessed. RESULTS: SCRV showed closest similarity to HRV82 with some divergence from the prototype. Amino acid differences were concentrated within predicted neutralization epitopes within VP2, VP3 and VP1. CONCLUSION: Sequence analyses and differences in cell culture growth characteristics suggest that this virus is a variant of HRV which has distinctive properties from its respective prototype strain.     

Mutations in the BC‐loop of the BKV VP1 region do not influence viral load in renal transplant patients

The reactivation and replication of the BK polyomavirus (BKV) leading to BKV‐associated nephropathy (BKVAN) is one of the major complications in renal transplantation patients. BKV isolates were classified into four subtypes (I‐IV) based on genotype variations within the VP1‐coding region. The type‐specific amino acid differences cluster within the BC‐loop of the major capsid protein VP1. As demonstrated in vitro, mutations in this region also play a role in the infectivity, attachment and stability of viral particles. Therefore, we analyzed the prevalence of BC‐loop mutations in isolates of kidney transplant patients and compared their viral load in the urine. The VP1 subtyping regions of BKV isolates obtained from urine samples of 45 renal transplant patients were sequenced. The phylogenetic analysis of these sequences revealed that subtype I (66.67%) is the most prevalent genotype. The remaining isolates belong to subtype IV (33.33%). A high frequency of changes to specific amino acids within the BC‐loop was identified among the BKV isolates from renal transplant patients. Patients with BKVAN exhibited a higher viral replication than patients without nephropathy. Although titers of isolates of subtype I were higher than titers of subtype IV isolates, the difference did not reach statistical significance. In addition, amino acid changes in the BC‐loop did not influence the viral load and the incidence of BKVAN. These in vivo results demonstrate that high replication rates which serve as a predictive marker for BKVAN are not caused by altered receptor binding or affinity via mutated BC‐loops. J. Med. Virol. 81:75–81, 2009. © 2008 Wiley‐Liss, Inc.     

Selection and characterization of an acid-resistant mutant of serotype O foot-and-mouth disease virus

Foot-and-mouth disease virus (FMDV) loses infectivity and immunogenicity due to its disassembly in culture environments below pH 6.8. To study the molecular basis of viral resistance to acid-induced disassembly and improve the acid stability of inactivated FMD vaccines during the manufacturing process, type O FMDV mutants with increased resistance to acid inactivation were selected, and the genes encoding their capsid proteins were sequenced. Three amino acid substitutions (VP1 N17D, VP2 D86A, and VP4 S73N) were found in all of the mutants. When these substitutions were introduced into seven infectious FMDV clones alone or combined, a single amino acid substitution in the VP1 protein, N17D, which also appears in type C FMDV acid-resistant mutants, was found to be responsible for the increased resistance to acid inactivation for type O FMDV. In addition, although viral fitness was reduced under standard culture conditions, viral growth kinetics and virulence were not significantly altered in the rescued mutant virus rN17D with the VP1 N17D substitution. Importantly, the N17D substitution could confer improved immunogenicity to the mutant virus rN17D under acidic conditions compared with its parental virus O/YS/CHA/05. These results demonstrate that the N17D substitution in VP1 is the molecular determinant of the acid-resistant phenotype in type O FMDV, indicating the potential for use of this substitution to improve the acid stability of inactivated FMD vaccines during the vaccine production process.     

Conformational changes in the hepatitis A virus capsid in response to acidic conditions

Low pH values encountered during uptake of viruses by receptor-mediated endocytosis have been shown to expose hydrophobic residues of many viruses and result in viral conformational changes leading to uncoating of the viral genome. An assay for hydrophobicity utilising the non-ionic detergent Triton X-114 was established, making use of metabolically-labelled hepatitis A virus (HAV). In this assay, hydrophilic proteins interact with the aqueous (buffer) phase, while hydrophobic proteins interact with the Triton (detergent) phase. HAV particles interact with the aqueous phase at neutral pH, whereas, under acidic conditions, HAV was found predominantly in the detergent phase. This indicates that the capsid of HAV undergoes conformational changes rendering the particle more hydrophobic under acidic conditions. A further two conformational changes were found in HAV on exposure to low pH, as detected by changes in buoyant density in CsCl gradients. These were maturation of provirions to virions and the formation of dense particles. These results may have implications for uncoating of the HAV RNA genome, and these conformational changes could represent intermediates in the viral uncoating process.     

The importance of inter- and intramolecular base pairing for translation reinitiation on a eukaryotic bicistronic mRNA

Calicivirus structure proteins are expressed from a subgenomic mRNA with two overlapping cistrons. The first ORF of this RNA codes for the viral major capsid protein VP1, and the second for the minor capsid protein VP2. Translation of VP2 is mediated by a termination/reinitiation mechanism, which depends on an upstream sequence element of ~70 nucleotides denoted “termination upstream ribosomal binding site” (TURBS). Two short sequence motifs within the TURBS were found to be essential for reinitiation. By a whole set of single site mutations and reciprocal base exchanges we demonstrate here for the first time conclusive evidence for the necessity of mRNA/18S rRNA hybridization for translation reinitiation in an eukaryotic system. Moreover, we show that motif 2 exhibits intramolecular hybridization with a complementary region upstream of motif 1, thus forming a secondary structure that positions post-termination ribosomes in an optimal distance to the VP2 start codon. Analysis of the essential elements of the TURBS led to a better understanding of the requirements for translation termination/reinitiation in eukaryotes.     

Second-site mutations encoding residues 34 and 78 of the major capsid protein (VP5) of herpes simplex virus type 1 are important for overcoming a blocked maturation cleavage site of the capsid scaffold proteins

During assembly of the herpes simplex type 1 capsid, the major capsid protein VP5 interacts with the C-terminal residues of the scaffold proteins encoded by UL26 and UL26.5. Subsequent to capsid assembly the scaffold proteins are cleaved at the maturation site by a serine protease also encoded by UL26, thereby enabling the bulk of the scaffold proteins to be released from the capsid. Previously, a mutant virus (KUL26-610/611) was isolated in which this maturation cleavage site was blocked by replacing the Ala/Ser at the 610/611 cleavage site by Glu/Phe. This mutation was lethal and required a transformed cell line expressing wild-type UL26 gene products for growth. Although the mutation was lethal, spontaneous reversions occurred at a high frequency. Previously, a small number of revertants were isolated and all were found to have second-site mutations in VP5. The purpose of the present study was to do a comprehensive determination of the sites altered in VP5 by the second-site mutations. To do this, an additional 25 independent spontaneous revertants were characterized. Seven of the 25 arose by GC --> GT changes in codon 78, giving rise to an alanine to valine substitution. Four were the result of base changes at codon 34 but two different amino acids were produced as the changes were at different positions in the codon. Two mutations were detected at position 41 and mutations that occurred once were found at codons 69 and 80. Thus, 15 of the 25 second-site mutants were localized to codons 34 to 80 of VP5, which contains 1374 amino acids. The remaining 10 revertants had codon changes at nine different sites, of which the most N-terminal was altered at codon 187 and the most C-terminal at codon 1317. As noted in the much smaller study a preponderance of the second-site mutants in VP5 were altered in codons at the extreme N-terminus of VP5. It is especially noteworthy that 11 out of 25 of the mutations occurred at codons 34 and 78. As expected, all of the revertants isolated were shown to retain the original KUL26-610/611 mutation, and the scaffold proteins remain uncleaved. All showed decreased retention of VP24 in the B capsids compared to the wild-type KOS, but more than the KUL26-610/611 parental virus. The revertants all had decreased growth rates of 2 to 18% compared to that of KOS and showed varying degrees of sensitivity when grown at 39.5 degrees C. The mutations in VP5 of three of the previously isolated viruses (PR5, PR6, and PR7) were transferred into a wild-type background, i.e., a virus encoding wild-type UL26 and UL26.5 gene products. All replicated in nonpermissive (Vero) cells and cleaved scaffold proteins. PR5 and PR6 in the wild-type background gave wild-type burst sizes and gave C-capsids that retained VP24 at approximately wild-type levels. The third revertant, PR7, in the wild-type background showed only a twofold increase of burst size (to 20% of wild-type) and the capsids showed little or no increase of VP24 retention. Therefore, the second-site mutations of PR7 (R69C) by itself had a negative effect on virus replication. By contrast the temperature sensitivity of PR6 and PR7 remained unchanged in the wild-type background. Thus the temperature sensitivity of PR6 and PR7 resides in VP5 independently of the mutation in the UL26 cleavage site.     

Antigens to viral capsid and non-capsid proteins are present in brain tissues and antibodies in sera of Theiler's virus-infected mice

Recombinant proteins to the LP, VP1, VP2, VP3, VP4, 2A, 2B, 2C, 3A, and 3D genes of Theiler's murine encephalomyelitis virus (TMEV) were generated and antibodies were produced against them for use in analysis of the TMEV epitopes responsible for eliciting the antibody responses observed during acute and chronic disease. Antibodies against recombinant VP1, VP2, and VP3 recognized the corresponding proteins from purified TMEV particles. In immunohistochemical analysis, antibodies against recombinant capsid (VP1, VP2, and VP3), and non-capsid (2A, 2C, 3A) proteins were reactive with PO-2D cells (astrocytes) infected with TMEV in vitro and with brain tissues of acutely infected mice. Antibodies against VP4, 2B, and 3D antigens were not reactive with corresponding viral proteins in infected astrocytes cells or brain tissues, but they reacted with TMEV precursor proteins produced during the early viral replication phase. Sera from SJL/J mice infected with TMEV acutely (14 days) and chronically (45 days) reacted with VP1, VP2, VP4, 2A, and 2C proteins. In an in vitro assay for neutralization, only anti-VP1 antibodies neutralized TMEV infection. These findings suggest that both capsid and non-capsid proteins of TMEV play a role in the immunopathology of the TMEV disease in the central nervous system.     

Genetic analysis of HAV strains in Tunisia reveals two new antigenic variants

To evaluate the genetic variability of hepatitis A virus (HAV) isolates in Tunisia, serum samples were collected from 99 patients in different Tunisian areas in 2003 containing 92 cases with acute hepatitis, five with severe acute hepatitis and two with fulminant hepatitis. The entire VP1 gene was amplified and sequenced. Sequences were then aligned and a phylogenetic analysis was performed. Additionally, the amino acid (aa) sequence of the VP1 was determined. The analysis of Tunisian HAV isolates revealed that all the isolates were sub-genotype IA with 96.4%–99.8% of identity and showed the emergence of two novel antigenic variants. The Tun31-03 antigenic variant, with a 38 aa deletion containing Met156, Val171, Leu174 and Ala176 and located between 150 and 187 aa of the VP1 protein where neutralization escape mutations, was found. The second antigenic variant, Tun36-03, was isolated from a patient with fulminant hepatitis and presented a substitution of Thr by Pro at position 10 of the VP1 protein. This amino acid is located in a peptide presenting an antigenically reactive epitope of the VP1 protein. This substitution has never been described previously.