IL-15 enhances CCR5-mediated migration of memory CD8+ T cells by upregulating CCR5 expression in the absence of TCR stimulation.

During microbial infection, bystander CD8+ T cells that are not specific to infecting pathogens can be activated by interleukin (IL)-15. However, the tissue-homing properties of bystander-activated CD8+ T cells have not been elucidated. Here, we examine the effects of IL-15 on the expression of chemokine receptors on CD8+ T cells and their migration. IL-15 upregulates CCR5 in memory CD8+ T cells in the absence of T cell receptor (TCR) stimulation and enhances CCR5-dependent migration. IL-15-induced CCR5 upregulation is abrogated by TCR stimulation, indicating that CCR5 is upregulated in bystander-activated CD8+ T cells. Moreover, CCR5 signals increase proliferation and cytotoxic protein expression in IL-15-treated memory CD8+ T cells, although the increase has a small extent. CCR5 upregulation in bystander-activated CD8+ T cells is associated with severe liver injury in patients with acute hepatitis A. Altogether, the results indicate that CCR5 upregulation by IL-15 mediates the migration of bystander-activated CD8+ T cells.

Use of Molecular Epidemiology to Inform Response to a Hepatitis A Outbreak - Los Angeles County, California, October 2018-April 2019.

Los Angeles County comprises 4,058 square miles and is home to approximately 10 million residents (1), an estimated 59,000 (0.6%) of whom experience homelessness on a given night (2). In late 2018, Los Angeles County Department of Public Health (LAC DPH) was notified of a case of hepatitis A virus (HAV) infection in a person experiencing homelessness. LAC DPH conducted an investigation to determine the source of infection, identify additional cases, and identify contacts for postexposure prophylaxis (PEP). Over the next week, LAC DPH identified two additional hepatitis A cases in persons experiencing homelessness who knew one another socially and were known to congregate at a specific street intersection. To identify and respond rapidly to additional outbreak-associated cases, LAC DPH implemented enhanced surveillance procedures, including immediately obtaining specimens for molecular testing from all patients with suspected hepatitis A in the same geographic area. Enhanced surveillance identified four additional cases in persons linked to a senior living campus within two blocks of the intersection where the initial three patients reported congregating. These four cases were linked to the cluster in persons experiencing homelessness through HAV genotyping. Overall, DPH identified seven outbreak-associated hepatitis A cases during October 2018-January 2019. The DPH response to this community hepatitis A outbreak included conducting vaccination outreach to persons at risk, conducting environmental health outreach to restaurants in the outbreak area, and issuing health care provider alerts about the increased occurrence of hepatitis A. Implementation of near real-time molecular testing can improve hepatitis A outbreak responses by confirming HAV infections, linking additional cases to the outbreak, and informing the targeting of prevention efforts.

Inherited IL-18BP deficiency in human fulminant viral hepatitis.

Fulminant viral hepatitis (FVH) is a devastating and unexplained condition that strikes otherwise healthy individuals during primary infection with common liver-tropic viruses. We report a child who died of FVH upon infection with hepatitis A virus (HAV) at age 11 yr and who was homozygous for a private 40-nucleotide deletion in IL18BP, which encodes the IL-18 binding protein (IL-18BP). This mutation is loss-of-function, unlike the variants found in a homozygous state in public databases. We show that human IL-18 and IL-18BP are both secreted mostly by hepatocytes and macrophages in the liver. Moreover, in the absence of IL-18BP, excessive NK cell activation by IL-18 results in uncontrolled killing of human hepatocytes in vitro. Inherited human IL-18BP deficiency thus underlies fulminant HAV hepatitis by unleashing IL-18. These findings provide proof-of-principle that FVH can be caused by single-gene inborn errors that selectively disrupt liver-specific immunity. They also show that human IL-18 is toxic to the liver and that IL-18BP is its antidote.

Redundant Late Domain Functions of Tandem VP2 YPX3L Motifs in Nonlytic Cellular Egress of Quasi-enveloped Hepatitis A Virus.

The quasi-envelopment of hepatitis A virus (HAV) capsids in exosome-like virions (eHAV) is an important but incompletely understood aspect of the hepatovirus life cycle. This process is driven by recruitment of newly assembled capsids to endosomal vesicles into which they bud to form multivesicular bodies with intraluminal vesicles that are later released at the plasma membrane as eHAV. The endosomal sorting complexes required for transport (ESCRT) are key to this process, as is the ESCRT-III-associated protein, ALIX, which also contributes to membrane budding of conventional enveloped viruses. YPX1or3L late domains in the structural proteins of these viruses mediate interactions with ALIX, and two such domains exist in the HAV VP2 capsid protein. Mutational studies of these domains are confounded by the fact that the Tyr residues (important for interactions of YPX1or3L peptides with ALIX) are required for efficient capsid assembly. However, single Leu-to-Ala substitutions within either VP2 YPX3L motif (L1-A and L2-A mutants) were well tolerated, albeit associated with significantly reduced eHAV release. In contrast, simultaneous substitutions in both motifs (L1,2-A) eliminated virus release but did not inhibit assembly of infectious intracellular particles. Immunoprecipitation experiments suggested that the loss of eHAV release was associated with a loss of ALIX recruitment. Collectively, these data indicate that HAV YPX3L motifs function as redundant late domains during quasi-envelopment and viral release. Since these motifs present little solvent-accessible area in the crystal structure of the naked extracellular capsid, the capsid structure may be substantially different during quasi-envelopment.IMPORTANCE Nonlytic release of hepatitis A virus (HAV) as exosome-like quasi-enveloped virions is a unique but incompletely understood aspect of the hepatovirus life cycle. Several lines of evidence indicate that the host protein ALIX is essential for this process. Tandem YPX3L "late domains" in the VP2 capsid protein could be sites of interaction with ALIX, but they are not accessible on the surface of an X-ray model of the extracellular capsid, raising doubts about this putative late domain function. Here, we describe YPX3L domain mutants that assemble capsids normally but fail to bind ALIX and be secreted as quasi-enveloped eHAV. Our data support late domain function for the VP2 YPX3L motifs and raise questions about the structure of the HAV capsid prior to and following quasi-envelopment.

Molecular characterization of hepatitis A virus from children hospitalized at a tertiary care centre in northwest India.

Background & objectives: Hepatitis A virus (HAV) infection is a major cause of childhood hepatitis, prevalent worldwide. HAV is classified into seven genotypes I-VII; genotypes III and I are the most common among humans. The present work was carried out to identify the genotypes prevalent in children suspected to have acute viral hepatitis (AVH), hospitalized at a tertiary care centre in northwest India. Methods: A total of 1269 blood samples from children (0-15 yr of age) clinically suspected of viral hepatitis were screened for anti-HAV IgM. Acute phase serum was processed for RNA extraction and amplified by nested polymerase chain reaction (PCR) followed by sequencing of representative samples. Results: Among the 1269 samples tested, 642 (50.59%) were positive for anti-HAV IgM; among the positive samples, 171 patients having a history of less than seven days were tested by PCR, of whom 141 (82.45%) were found to be PCR positive. Nucleotide sequencing of a representative 44 samples showed high homology; all the samples were found to be of genotype IIIA. Interpretation & conclusions: Hepatitis A was prevalent during July to September and in predominantly children less than five years age. Only genotype IIIA was detected in all the samples.

Relationship of Severity of Hepatitis A with Polymorphisms in Hepatitis A Virus Cellular Receptor 1 (HAVCR1) Gene.

INTRODUCTION AND AIM: HAVCR1 protein is the cellular receptor for hepatitis A virus (HAV). Genetic polymorphism in this gene may alter the outcome of HAV infection. In a previous study, a 6-amino acid insertion (157insMTTTVP) in HAVCR1 gene was associated with more severe disease. We decided to investigate this association further. MATERIAL AND METHODS: We sequenced exon 4 of the HAVCR1 gene in patients with clinical hepatitis A attending our institution, and a group of healthy controls in a disease-endemic setting in India. Frequencies of different haplotypes of a genomic region with two overlapping insertion-deletion polymorphisms (indels; rs141023871 and rs139041445) were compared between patients and controls, as well as between patients with and without a severe form of disease (liver failure). RESULTS: The gene had three haplotypes in the region of interest - a short form, an intermediate-form with a 5-amino acid 157insMTTVP insertion and a long-form with a 6-amino acid 157insMTTTVP insertion. The allele frequency (29/150 [19%] vs. 43/146 [29%]; p = ns) and haplotype frequency (29/75 [39%] vs. 39/73 [53%]; p = ns) of the 157insMTTTVP variant were similar in hepatitis A patients and healthy controls (30%). Further, the allele frequency (12/58 [21%] vs. 17/92 [18%]; p = ns) and haplotype frequency (12/29 [41%] vs.17/46 [37%]; p = ns) of the longest variant were also similar in patients with severe and mild disease. DISCUSSION: In the study population, the 157insMTTTVP variant of HAVCR1 gene was not associated with more severe outcome of HAV infection. Further studies in other populations around the world are needed to assess the relation of this genetic variation with disease outcome.

Evaluation of candidate genes associated with hepatitis A and E virus infection in Chinese Han population.

BACKGROUND: Recent GWAS-associated studies reported that single nucleotide polymorphisms (SNPs) in ABCB1, TGFß1, XRCC1 genes were associated with hepatitis A virus (HAV) infection, and variants of APOA4 and APOE genes were associated with and hepatitis E virus (HEV) infection in US population. However, the associations of these loci with HAV or HEV infection in Chinese Han population remain unclear. METHODS: A total of 3082 Chinese Han persons were included in this study. Anti-HAV IgG and anti-HEV IgG were detected by enzyme-linked immunosorbent assay (ELISA). Genotypes in ABCB1, TGFß1, XRCC1, APOA4 and APOE SNPs were determined by TaqMan MGB technology. RESULTS: In Chinese Han population, rs1045642 C to T variation in ABCB1 was significantly associated with the decreased risk of HAV infection (P < 0.05). However, the effect direction was different with the previous US study. Rs1001581 A to G variation in XRCC1, which was not identified in US population, was significantly associated with the protection against HAV infection in our samples (P < 0.05). In addition, our results suggested that rs7412 C to T variation in APOE was significantly associated with lower risk of HEV infection in males (adjusted OR < 1.0, P < 0.05) but not in females. CONCLUSIONS: ABCB1 and XRCC1 genes variants are significantly associated with the protection against HAV infection. Additionally, Chinese Han males with rs7412 C to T variation in APOE gene are less prone to be infected by HEV.

Genome-wide association and HLA region fine-mapping studies identify susceptibility loci for multiple common infections.

Infectious diseases have a profound impact on our health and many studies suggest that host genetics play a major role in the pathogenesis of most of them. We perform 23 genome-wide association studies for common infections and infection-associated procedures, including chickenpox, shingles, cold sores, mononucleosis, mumps, hepatitis B, plantar warts, positive tuberculosis test results, strep throat, scarlet fever, pneumonia, bacterial meningitis, yeast infections, urinary tract infections, tonsillectomy, childhood ear infections, myringotomy, measles, hepatitis A, rheumatic fever, common colds, rubella and chronic sinus infection, in over 200,000 individuals of European ancestry. We detect 59 genome-wide significant (P < 5 × 10-8) associations in genes with key roles in immunity and embryonic development. We apply fine-mapping analysis to dissect associations in the human leukocyte antigen region, which suggests important roles of specific amino acid polymorphisms in the antigen-binding clefts. Our findings provide an important step toward dissecting the host genetic architecture of response to common infections.Susceptibility to infectious diseases is, among others, influenced by the genetic landscape of the host. Here, Tian and colleagues perform genome-wide association studies for 23 common infections and find 59 risk loci for 17 of these, both within the HLA region and non-HLA loci.

CXCL10 is produced in hepatitis A virus-infected cells in an IRF3-dependent but IFN-independent manner.

Acute hepatitis A caused by hepatitis A virus (HAV) infection is accompanied by severe liver injury in adult patients, and the liver injury is associated with the production of chemokines. Herein, we investigated the mechanism of how HAV infection induces the production of CXCR3 and CCR5 chemokines, such as CXCL10, CCL4 and CCL5. The production of CXCL10, CCL4 and CCL5 was markedly increased by HAV (HM-175/18f) infection in the culture of primary human hepatocytes and HepG2 cells. In particular, CXCL10 was produced in HAV-infected cells, not in neighboring uninfected cells. Moreover, these chemokines were significantly increased in the sera of acute hepatitis A patients. The production of IFN-λs was also robustly induced by HAV infection, and the blocking of secreted IFN-λs partially abrogated the production of CCL4 and CCL5 in HAV-infected cells. However, CXCL10 production was not decreased by the blocking of IFN-λs. Instead, CXCL10 production was reduced by silencing the expression of RIG-I-like receptor (RLR) signal molecules, such as mitochondrial antiviral signaling protein and interferon regulatory factor 3, in HAV-infected cells. In conclusion, HAV infection strongly induces the production of helper 1 T cell-associated chemokines, particularly CXCL10 via RLR signaling, even without secreted IFNs.

Association between MFN2 gene polymorphisms and the risk and prognosis of acute liver failure: a case-control study in a Chinese population.

This study aimed to determine the role of mitofusin 2 (MFN2) gene polymorphisms in the risk and prognosis of acute liver failure (ALF). A total of 298 blood samples were collected from 138 ALF patients (case group) and 160 healthy participants (control group). Coagulation function, glutamic pyruvic transaminase (GPT), glutamic oxaloacetic transaminase (GOT), total bilirubin (TB), blood ammonia and lactic acid (LA) were measured. The predictive evaluation of MFN2 gene polymorphisms in the risk and prognosis of ALF patients was estimated using Kaplan-Meier survival analysis, haplotype analysis, binary logistic regression analysis and Cox regression analysis. Higher levels of GPT, GOT, TB, blood ammonia and LA were observed in ALF patients with the GG genotype of rs873457 or the TT genotype of rs4846085 than in those with the CC genotype of these two SNPs. The GTACAGC and GTGTGGC haplotypes were a protective factor and a risk factor for ALF, respectively. Blood ammonia and LA levels were independent risk factors and the CC genotype of rs873457 and the CC genotype of rs4846085 were protective factors for ALF. ALF patients with the GG genotype of rs873457 or the TT genotype of rs4846085 had a lower survival rate than those with other genotypes of these two SNPs. The rs4846085 and rs873457 polymorphisms were both independent factors affecting the prognosis of ALF patients. MFN2 gene polymorphisms (rs873457, rs2336384, rs1474868, rs4846085 and rs2236055) may be associated with ALF and the rs873457 and rs4846085 polymorphisms are correlated with the risk and prognosis of ALF.

Association between MFN2 gene polymorphisms and the risk and prognosis of acute liver failure: a case-control study in a Chinese population

This study aimed to determine the role of mitofusin 2 (MFN2) gene polymorphisms in the risk and prognosis of acute liver failure (ALF). A total of 298 blood samples were collected from 138 ALF patients (case group) and 160 healthy participants (control group). Coagulation function, glutamic pyruvic transaminase (GPT), glutamic oxaloacetic transaminase (GOT), total bilirubin (TB), blood ammonia and lactic acid (LA) were measured. The predictive evaluation of MFN2 gene polymorphisms in the risk and prognosis of ALF patients was estimated using Kaplan-Meier survival analysis, haplotype analysis, binary logistic regression analysis and Cox regression analysis. Higher levels of GPT, GOT, TB, blood ammonia and LA were observed in ALF patients with the GG genotype of rs873457 or the TT genotype of rs4846085 than in those with the CC genotype of these two SNPs. The GTACAGC and GTGTGGC haplotypes were a protective factor and a risk factor for ALF, respectively. Blood ammonia and LA levels were independent risk factors and the CC genotype of rs873457 and the CC genotype of rs4846085 were protective factors for ALF. ALF patients with the GG genotype of rs873457 or the TT genotype of rs4846085 had a lower survival rate than those with other genotypes of these two SNPs. The rs4846085 and rs873457 polymorphisms were both independent factors affecting the prognosis of ALF patients. MFN2 gene polymorphisms (rs873457, rs2336384, rs1474868, rs4846085 and rs2236055) may be associated with ALF and the rs873457 and rs4846085 polymorphisms are correlated with the risk and prognosis of ALF.

Comprehensive profiling and characterization of cellular miRNAs in response to hepatitis A virus infection in human fibroblasts.

Hepatitis A virus (HAV), the causative agent of acute hepatitis, grows slowly without causing any cytopathic effect (CPE) and lead to a persistent infection in the fibroblasts in vitro. miRNAs play a key role in the viral pathogenesis and virus-host interactions. In this study, the comprehensive miRNA expression profiles of HAV-infected and uninfected fibroblasts were investigated by sRNA-seq and validated by RT-qPCR. The results showed that a total of 94 miRNAs were differentially expressed during HAV infection, including 11 up-regulated miRNAs and 83 down-regulated miRNAs. RT-qPCR analysis showed the expression levels of specific miRNAs were consistent with sRNA-seq data. Further, target prediction analysis showed 729 putative target genes that included many immune-related transcripts were revealed. The GO enrichment analysis and the KEGG pathway analysis of the target genes showed that various biological pathways, including JAK-STAT cascade, type I interferon signaling pathway could be affected by HAV infection by the alteration of host miRNAs. The core regulatory relationship between miRNAs and their targets were revealed by miRNA-gene-network. Collectively, this study provides an overall analysis of miRNA profile in cell culture infected with HAV. The present results imply the alteration of miRNAs expression induced by HAV infection which may be related to the establishment of persistent HAV infection and might provide new clues for understanding the persistent HAV infections in vitro and the unique biological characteristics associated with HAV during infection.

Sandwich Immunoassays of Multicomponent Subtrace Pathogenic DNA Based on Magnetic Fluorescent Encoded Nanoparticles.

A novel magnetic fluorescent encoded nanoimmunoassay system for multicomponent detection and separation of the subtrace pathogenic DNA (hepatitis B virus surface gene, HBV; hepatitis A virus poly the protein gene, HAV) was established based on new type of magnetic fluorescent encoded nanoparticles and sandwich immunoassay principle. This method combines multifunctional nanoparticles, immunoassay technique, fluorescence labeling, and magnetic separation of multicomponent technology. It has many advantages such as high sensitivity, low detection limit, easy operation, and great potential for development. The results of this work show that, based on nanoimmunoassay system, it could quantitatively detect the multicomponent trace pathogenic HAV and HBV DNA, as well as detection limit up to 0.1 pM and 0.12 pM. Furthermore, with the improvement of the performances of magnetic fluorescent encoded nanoparticles, the sensitivity will be further improved. In this experiment, a new nanoimmunoassay system based on magnetic fluorescent encoded nanoparticles was established, which will provide a new way for the immunoassay and separation of multicomponent biomolecules.

Micro RNAs mir-106a, mir-122 and mir-197 are increased in severe acute viral hepatitis with coagulopathy.

BACKGROUND & AIMS: The severity of acute viral hepatitis, which may be caused by several distinct viruses, varies among individual patients. In rare cases, severe hepatic injury with sudden loss of liver function may occur, which is clinically indicated by the occurrence of coagulopathy or encephalopathy. As the molecular mechanisms of this liver injury are largely unknown, we investigated extracellular micro RNA (miRNA) profiles in 54 patients acutely infected with one of four different hepatotropic viruses, in order to identify those miRNAs which indicate severe viral hepatitis associated with coagulopathy. METHODS: First, the profile of miRNAs was extensively analysed using a microarray-based approach in highly characterized 24 patients, matched in terms of sex, age and level of liver enzymes, as well as in three healthy controls. The cohort included samples from 18 patients with moderate and six individuals with severe hepatitis, indicated by abnormal prothrombin time and higher alanine aminotransferase and bilirubin levels. miRNAs found to be upregulated in severe hepatitis were then quantified by real-time PCR in the expanded cohort of 54 patients. RESULTS: Comprehensive microarray-based miRNA profiling identified upregulation of mir-106a, mir-122 and mir-197 in patients with severe acute viral hepatitis with coagulopathy, as compared to patients who did not develop coagulopathy. mir-106a, mir-122 and mir-197 were then proven to be significantly upregulated in patients with severe acute viral hepatitis by quantitative real-time PCR (P < 0.01, Mann-Whitney U-test). CONCLUSIONS: mir-106a, mir-122 and mir-197 could be potential markers for severe acute viral hepatitis associated with coagulopathy.

Phenotypic Characteristics of PD-1 and CTLA-4 Expression in Symptomatic Acute Hepatitis A.

BACKGROUND/AIMS: The immunoregulatory molecules programmed death 1 (PD-1) and cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) are associated with the dysfunction of antiviral effector T-cells, which leads to T-cell exhaustion and persistent viral infection in patients with chronic hepatitis C and chronic hepatitis B. Little is known about the role of PD-1 and CTLA-4 in patients with symptomatic acute hepatitis A (AHA). METHODS: Peripheral blood mononuclear cells were isolated from seven patients with AHA and from six patients with nonviral acute toxic hepatitis (ATH) during the symptomatic and convalescent phases of the respective diseases; five healthy subjects acted as controls. The expression of PD-1 and CTLA-4 on T-cells was measured by flow cytometry. RESULTS: PD-1 and CTLA-4 expression during the symptomatic phase was significantly higher in the T-cells of AHA patients than in those of ATH patients or healthy controls (PD-1 18.3% vs 3.7% vs 1.6%, respectively, p<0.05; CTLA-4 23.5% vs 6.1% vs 5.9%, respectively, p<0.05). The levels of both molecules decreased dramatically during the convalescent phase of AHA, whereas a similar pattern was not seen in ATH. CONCLUSIONS: Our findings are consistent with a viral-protective effect of PD-1 and CTLA-4 as inhibitory molecules that suppress cytotoxic T-cells and thereby prevent the destruction of virus-infected hepatocytes in AHA.

Phenotypic Characteristics of PD-1 and CTLA-4 Expression in Symptomatic Acute Hepatitis A

BACKGROUND/AIMS: The immunoregulatory molecules programmed death 1 (PD-1) and cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) are associated with the dysfunction of antiviral effector T-cells, which leads to T-cell exhaustion and persistent viral infection in patients with chronic hepatitis C and chronic hepatitis B. Little is known about the role of PD-1 and CTLA-4 in patients with symptomatic acute hepatitis A (AHA). METHODS: Peripheral blood mononuclear cells were isolated from seven patients with AHA and from six patients with nonviral acute toxic hepatitis (ATH) during the symptomatic and convalescent phases of the respective diseases; five healthy subjects acted as controls. The expression of PD-1 and CTLA-4 on T-cells was measured by flow cytometry. RESULTS: PD-1 and CTLA-4 expression during the symptomatic phase was significantly higher in the T-cells of AHA patients than in those of ATH patients or healthy controls (PD-1: 18.3% vs 3.7% vs 1.6%, respectively, p<0.05; CTLA-4: 23.5% vs 6.1% vs 5.9%, respectively, p<0.05). The levels of both molecules decreased dramatically during the convalescent phase of AHA, whereas a similar pattern was not seen in ATH. CONCLUSIONS: Our findings are consistent with a viral-protective effect of PD-1 and CTLA-4 as inhibitory molecules that suppress cytotoxic T-cells and thereby prevent the destruction of virus-infected hepatocytes in AHA.

Molecular characterization of hepatitis A virus strains in a tertiary care health set up in north western India.

BACKGROUND & OBJECTIVES: Hepatitis A virus usually causes acute viral hepatitis (AVH) in the paediatric age group with a recent shift in age distribution and disease manifestations like acute liver failure (ALF). This has been attributed to mutations in 5'non-translated region (5'NTR) which affects the viral multiplication. The present study was aimed to carry out the molecular detection and phylogenetic analysis of hepatitis A virus strains circulating in north western India. METHODS: Serum samples from in patients and those attending out patient department of Pediatric Gastroenterology in a tertiary care hospital in north India during 2007-2011 with clinically suspected AVH were tested for anti-hepatitis A virus (HAV) IgM antibodies. Acute phase serum samples were subjected to nested PCR targeting the 5'NTR region followed by sequencing of the representative strains. RESULTS: A total of 1334 samples were tested, 290 (21.7%) were positive for anti-HAV IgM antibody. Of these, 78 serum samples (< 7 days old) were subjected to PCR and 47.4% (37/78) samples showed the presence of HAV RNA. Children < 15 yr of age accounted for majority (94%) of cases with highest seropositivity during rainy season. Sequencing of 15 representative strains was carried out and the circulating genotype was found to be III A. The nucleotide sequences showed high homology among the strains with a variation ranging from 0.1-1 per cent over the years. An important substitution of G to A at 324 position was shown by both AVH and ALF strains. The cumulative substitution in AVH strains Vs ALF strains as compared to GBM, Indian and prototype strain in the 200-500 region of 5' NTR was comparable. INTERPRETATION & CONCLUSION: Our results showed hepatitis A still a disease of children with III A as a circulating genotype in this region. The mutations at 5'NTR region warrant further analysis as these affect the structure of internal ribosomal entry site which is important for viral replication.

Identification of a conserved B-cell epitope on duck hepatitis A type 1 virus VP1 protein.

BACKGROUND: The VP1 protein of duck hepatitis A virus (DHAV) is a major structural protein that induces neutralizing antibodies in ducks; however, B-cell epitopes on the VP1 protein of duck hepatitis A genotype 1 virus (DHAV-1) have not been characterized. METHODS AND RESULTS: To characterize B-cell epitopes on VP1, we used the monoclonal antibody (mAb) 2D10 against Escherichia coli-expressed VP1 of DHAV-1. In vitro, mAb 2D10 neutralized DHAV-1 virus. By using an array of overlapping 12-mer peptides, we found that mAb 2D10 recognized phages displaying peptides with the consensus motif LPAPTS. Sequence alignment showed that the epitope 173LPAPTS178 is highly conserved among the DHAV-1 genotypes. Moreover, the six amino acid peptide LPAPTS was proven to be the minimal unit of the epitope with maximal binding activity to mAb 2D10. DHAV-1-positive duck serum reacted with the epitope in dot blotting assay, revealing the importance of the six amino acids of the epitope for antibody-epitope binding. Competitive inhibition assays of mAb 2D10 binding to synthetic LPAPTS peptides and truncated VP1 protein fragments, detected by Western blotting, also verify that LPAPTS was the VP1 epitope. CONCLUSIONS AND SIGNIFICANCE: We identified LPAPTS as a VP1-specific linear B-cell epitope recognized by the neutralizing mAb 2D10. Our findings have potential applications in the development of diagnostic techniques and epitope-based marker vaccines against DHAV-1.