Biochemical analysis of the host factor activity of ZCCHC14 in hepatitis A virus replication.

Relatively little is known of the mechanisms underlying hepatitis A virus (HAV) genome replication. Unlike other well-studied picornaviruses, HAV RNA replication requires the zinc finger protein ZCCHC14 and non-canonical TENT4 poly(A) polymerases with which it forms a complex. The ZCCHC14-TENT4 complex binds to a stem-loop located within the internal ribosome entry site (IRES) in the 5' untranslated RNA (5'UTR) and is essential for viral RNA synthesis, but the underlying mechanism is unknown. Here, we describe how different ZCCHC14 domains contribute to its RNA-binding, TENT4-binding, and HAV host factor activities. We show that the RNA-binding activity of ZCCHC14 requires both a sterile alpha motif (SAM) and a downstream unstructured domain (D4) and that ZCCHC14 contains two TENT4-binding sites: one at the N-terminus and the other around D4. Both RNA-binding and TENT4-binding are required for HAV host factor activity of ZCCHC14. We also demonstrate that the location of the ZCCHC14-binding site within the 5'UTR is critical for its function. Our study provides a novel insight into the function of ZCCHC14 and helps elucidate the mechanism of the ZCCHC14-TENT4 complex in HAV replication.IMPORTANCEThe zinc finger protein ZCCHC14 is an essential host factor for both hepatitis A virus (HAV) and hepatitis B virus (HBV). It recruits the non-canonical TENT4 poly(A) polymerases to viral RNAs and most likely also a subset of cellular mRNAs. Little is known about the details of these interactions. We show here the functional domains of ZCCHC14 that are involved in binding to HAV RNA and interactions with TENT4 and describe previously unrecognized peptide sequences that are critical for the HAV host factor activity of ZCCHC14. Our study advances the understanding of the ZCCHC14-TENT4 complex and how it functions in regulating viral and cellular RNAs.

The ZCCHC14/TENT4 complex is required for hepatitis A virus RNA synthesis.

Despite excellent vaccines, resurgent outbreaks of hepatitis A have caused thousands of hospitalizations and hundreds of deaths within the United States in recent years. There is no effective antiviral therapy for hepatitis A, and many aspects of the hepatitis A virus (HAV) replication cycle remain to be elucidated. Replication requires the zinc finger protein ZCCHC14 and noncanonical TENT4 poly(A) polymerases with which it associates, but the underlying mechanism is unknown. Here, we show that ZCCHC14 and TENT4A/B are required for viral RNA synthesis following translation of the viral genome in infected cells. Cross-linking immunoprecipitation sequencing (CLIP-seq) experiments revealed that ZCCHC14 binds a small stem-loop in the HAV 5' untranslated RNA possessing a Smaug recognition-like pentaloop to which it recruits TENT4. TENT4 polymerases lengthen and stabilize the 3' poly(A) tails of some cellular and viral mRNAs, but the chemical inhibition of TENT4A/B with the dihydroquinolizinone RG7834 had no impact on the length of the HAV 3' poly(A) tail, stability of HAV RNA, or cap-independent translation of the viral genome. By contrast, RG7834 inhibited the incorporation of 5-ethynyl uridine into nascent HAV RNA, indicating that TENT4A/B function in viral RNA synthesis. Consistent with potent in vitro antiviral activity against HAV (IC50 6.11 nM), orally administered RG7834 completely blocked HAV infection in Ifnar1-/- mice, and sharply reduced serum alanine aminotransferase activities, hepatocyte apoptosis, and intrahepatic inflammatory cell infiltrates in mice with acute hepatitis A. These results reveal requirements for ZCCHC14-TENT4A/B in hepatovirus RNA synthesis, and suggest that TENT4A/B inhibitors may be useful for preventing or treating hepatitis A in humans.

IRF3-mediated pathogenicity in a murine model of human hepatitis A.

HAV-infected Ifnar1-/- mice recapitulate many of the cardinal features of hepatitis A in humans, including serum alanine aminotransferase (ALT) elevation, hepatocellular apoptosis, and liver inflammation. Previous studies implicate MAVS-IRF3 signaling in pathogenesis, but leave unresolved the role of IRF3-mediated transcription versus the non-transcriptional, pro-apoptotic activity of ubiquitylated IRF3. Here, we compare the intrahepatic transcriptomes of infected versus naïve Mavs-/- and Ifnar1-/- mice using high-throughput sequencing, and identify IRF3-mediated transcriptional responses associated with hepatocyte apoptosis and liver inflammation. Infection was transcriptionally silent in Mavs-/- mice, in which HAV replicates robustly within the liver without inducing inflammation or hepatocellular apoptosis. By contrast, infection resulted in the upregulation of hundreds of genes in Ifnar1-/- mice that develop acute hepatitis closely modeling human disease. Upregulated genes included pattern recognition receptors, interferons, chemokines, cytokines and other interferon-stimulated genes. Compared with Ifnar1-/- mice, HAV-induced inflammation was markedly attenuated and there were few apoptotic hepatocytes in livers of infected Irf3S1/S1Ifnar1-/- mice in which IRF3 is transcriptionally-inactive due to alanine substitutions at Ser-388 and Ser-390. Although transcriptome profiling revealed remarkably similar sets of genes induced in Irf3S1/S1Ifnar1-/- and Ifnar1-/- mice, a subset of genes was differentially expressed in relation to the severity of the liver injury. Prominent among these were both type 1 and type III interferons and interferon-responsive genes associated previously with apoptosis, including multiple members of the ISG12 and 2'-5' oligoadenylate synthetase families. Ifnl3 and Ifnl2 transcript abundance correlated strongly with disease severity, but mice with dual type 1 and type III interferon receptor deficiency remained fully susceptible to liver injury. Collectively, our data show that IRF3-mediated transcription is required for HAV-induced liver injury in mice and identify key IRF3-responsive genes associated with pathogenicity, providing a clear distinction from the transcription-independent role of IRF3 in liver injury following binge exposure to alcohol.

Exosome mimicry by a HAVCR1-NPC1 pathway of endosomal fusion mediates hepatitis A virus infection.

Cell-to-cell communication by exosomes controls normal and pathogenic processes1,2. Viruses can spread in exosomes and thereby avoid immune recognition3. While biogenesis, binding and uptake of exosomes are well characterized4,5, delivery of exosome cargo into the cytoplasm is poorly understood3. We report that the phosphatidylserine receptor HAVCR1 (refs. 6,7) and the cholesterol transporter NPC1 (ref. 8) participate in cargo delivery from exosomes of hepatitis A virus (HAV)-infected cells (exo-HAV) by clathrin-mediated endocytosis. Using CRISPR-Cas9 knockout technology, we show that these two lipid receptors, which interact in the late endosome9, are necessary for the membrane fusion and delivery of RNA from exo-HAV into the cytoplasm. The HAVCR1-NPC1 pathway, which Ebola virus exploits to infect cells9, mediates HAV infection by exo-HAV, which indicates that viral infection via this exosome mimicry mechanism does not require an envelope glycoprotein. The capsid-free viral RNA in the exosome lumen, but not the endosomal uncoating of HAV particles contained in the exosomes, is mainly responsible for exo-HAV infectivity as assessed by methylene blue inactivation of non-encapsidated RNA. In contrast to exo-HAV, infectivity of HAV particles is pH-independent and requires HAVCR1 or another as yet unidentified receptor(s) but not NPC1. Our findings show that envelope-glycoprotein-independent fusion mechanisms are shared by exosomes and viruses, and call for a reassessment of the role of envelope glycoproteins in infection.

Gender Impacts on the Disease Severity of Overt Acute Hepatitis A: Different from Overt Acute Hepatitis B.

BACKGROUND: Acute hepatitis A (AH-A) and acute hepatitis B (AH-B) were found more severe in males and females, respectively, while impacts from underlying liver disease on severity were not excluded in the AH-A study. AIM: The precise gender-specific impact on the severity of AH-A was investigated and compared with that of AH-B. METHODS: A case-control study of overt AH-A (n = 118) and AH-B (n = 118) patients without any underlying liver disease was conducted. Overt hepatitis was defined as serum bilirubin ≥ 2 mg/dL and alanine transaminase (ALT) ≥ 10 × upper limit of normal. RESULTS: Of the AH-A patients, age (95% confidence interval of odds ratio 1.051-1.147) and ALT (1.001-1.002) were associated with hepatic decompensation. Indifferent rates of hepatic decompensation, hepatic failure, and mortality were found between male and female patients. Compared with the AH-B patients, AH-A patients showed lower bilirubin levels (p < 0.001), hepatic decompensation (p = 0.004), and mortality rates (p = 0.013). Among patients < 40 years, the AH-A patients had higher hepatic decompensation rates than AH-B in the male subgroup (15% vs. 2.8%, p = 0.045), while the situation is reverse in the female subgroup (7.7% vs. 48.1%, p = 0.001). CONCLUSIONS: Overt AH-A was less severe than overt AH-B and, unlike AH-B, had no difference in severity between males and females. Among subgroups < 40 years, AH-A was more severe than AH-B in males, but the situation was reverse in females in terms of hepatic decompensation rates.

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.

Association of Toll-like receptor 4 with hepatitis A virus infection in Assam.

Hepatitis A virus (HAV) which causes liver disease is recognized by Toll-like receptors (TLRs) through the viral nucleic acid, initiating the host defense response. The study aims to analyze the role of TLR4 rs11536889 polymorphism in the pathogenesis of hepatitis A cases from Assam. There was significant correlation between TLR4 SNP G/C (rs11536889) and between acute viral hepatitis (AVH) A cases and controls. The correlation of the 3 different genotypes GG, GC and CC of TLR4 rs11536889 with the TLR4 mRNA expression level in all the HAV cases groups have been found to be statistically significant (p <0.001). TLR4 expression was most significantly upregulated in the acute HAV cases, HAV with cholestasis cases and even the HAV caused fulminant hepatitis failure (FHF) cases with the CC genotype of TLR4 rs11536889. The upregulation is mostly seen in the cases with the CC genotype of TLR4 rs11536889 and thus indicates that the mutant variant of TLR4 rs11536899 (CC) may have an effect on the expression of TLR4 at the transcription level. Our study did not show any significant association between AVH and HAV caused FHF (p = 0.32, OR = 0; p = 0.59, OR = 2.06 at 95% CI) among the genotypes GG, GC and CC. Our data suggest that TLR4 gene polymorphism rs11536889 may play a prominent role in HAV disease susceptibility and TLR4 expression in population from Assam.

Tumor Necrosis Factor-producing T-regulatory Cells Are Associated With Severe Liver Injury in Patients With Acute Hepatitis A.

BACKGROUND AND AIMS: CD4+CD25+Foxp3+ T-regulatory (Treg) cells control immune responses and maintain immune homeostasis. However, under inflammatory conditions, Treg cells produce cytokines that promote inflammation. We investigated production of tumor necrosis factor (TNF) by Treg cells in patients with acute hepatitis A (AHA), and examined the characteristics of these cells and association with clinical factors. METHODS: We analyzed blood samples collected from 63 patients with AHA at the time of hospitalization (and some at later time points) and 19 healthy donors in South Korea. Liver tissues were collected from patients with fulminant AHA during liver transplantation. Peripheral blood mononuclear cells were isolated from whole blood and lymphocytes were isolated from liver tissues and analyzed by flow cytometry. Cytokine production from Treg cells (CD4+CD25+Foxp3+) was measured by immunofluorescence levels following stimulation with anti-CD3 and anti-CD28. Epigenetic stability of Treg cells was determined based on DNA methylation patterns. Phenotypes of Treg cells were analyzed by flow cytometry and an RORγt inhibitor, ML-209, was used to inhibit TNF production. Treg cell suppression assay was performed by co-culture of Treg-depleted peripheral blood mononuclear cells s and isolated Treg cells. RESULTS: A higher proportion of CD4+CD25+Foxp3+ Treg cells from patients with AHA compared with controls produced TNF upon stimulation with anti-CD3 and anti-CD28 (11.2% vs 2.8%). DNA methylation analysis confirmed the identity of the Treg cells. TNF-producing Treg cells had features of T-helper 17 cells, including up-regulation of RORγt, which was required for TNF production. The Treg cells had reduced suppressive functions compared with Treg cells from controls. The frequency of TNF-producing Treg cells in AHA patients' blood correlated with their serum level of alanine aminotransferase. CONCLUSIONS: Treg cells from patients with AHA have altered functions compared with Treg cells from healthy individuals. Treg cells from patients with AHA produce higher levels of TNF, gain features of T-helper 17 cells, and have reduced suppressive activity. The presence of these cells is associated with severe liver injury in patients with AHA.

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.

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.

Hepatitis A virus-encoded miRNAs attenuate the accumulation of viral genomic RNAs in infected cells.

The establishment of persistent infection with hepatitis A virus (HAV) is the common result of most HAV/cell culture systems. Previous observations show that the synthesis of viral RNAs is reduced during infection. However, the underlying mechanism is poorly understood. We characterized three HAV-encoded miRNAs in our previous study. In this study, we aim to investigate the impact of these miRNAs on the accumulation of viral RNAs. The results indicated that the synthesis of viral genomic RNAs was dramatically reduced (more than 75 % reduction, P < 0.05) when transfected with one or two viral miRNA mimics. Conversely, they were significantly increased (more than 3.3-fold addition, P < 0.05) when transfected with one or two viral miRNA inhibitors. The luciferase reporter assay of miRNA targets showed that viral miRNAs were fully complementary to specific sites of the viral plus or minus strand RNA and strongly inhibited their expressions. Further data showed that the relative abundance of viral genomic RNA fragments that contain miRNA targets was also dramatically reduced (more than 80 % reduction, P < 0.05) when viral miRNAs were overexpressed with miRNA mimics. In contrast, they were significantly increased (approximately 2-fold addition, P < 0.05) when viral miRNAs were inhibited with miRNA inhibitors. In conclusion, these data suggest a possible mechanism for the reduction of viral RNA synthesis during HAV infection. Thus, we propose that it is likely that RNA virus-derived miRNA could serve as a self-mediated feedback regulator during infection.

The JAK2 inhibitor AZD1480 inhibits hepatitis A virus replication in Huh7 cells.

The JAK2 inhibitor AZD1480 has been reported to inhibit La protein expression. We previously demonstrated that the inhibition of La expression could inhibit hepatitis A virus (HAV) internal ribosomal entry-site (IRES)-mediated translation and HAV replication in vitro. In this study, we analyzed the effects of AZD1480 on HAV IRES-mediated translation and replication. HAV IRES-mediated translation in COS7-HAV-IRES cells was inhibited by 0.1-1 µM AZD1480, a dosage that did not affect cell viability. Results showed a significant reduction in intracellular HAV HA11-1299 genotype IIIA RNA levels in Huh7 cells treated with AZD1480. Furthermore, AZD1480 inhibited the expression of phosphorylated-(Tyr-705)-signal transducer and activator of transcription 3 (STAT3) and La in Huh7 cells. Therefore, we propose that AZD1480 can inhibit HAV IRES activity and HAV replication through the inhibition of the La protein.

Monitoring liver macrophages using nanobodies targeting Vsig4: concanavalin A induced acute hepatitis as paradigm.

Kupffer cells (KCs) are liver resident macrophages which are important for tissue homeostasis and have been implicated in immunogenic, tolerogenic and pathogenic immune reactions depending on the insult. These cells and the biomarkers they express thus represent interesting in vivo sensors for monitoring liver inflammation. In the current study, we explored whether KCs can be monitored non-invasively using single-photon-emission computed tomography (SPECT) with (99m)Tc labeled nanobodies (Nbs) targeting selected biomarkers. Nbs targeting V-set and immunoglobulin domain-containing 4 (Vsig4) or macrophage mannose receptor (MMR) accumulated in the liver of untreated mice. The liver targeting of anti-Vsig4 Nbs, but not anti-MMR Nbs, was blunted upon depletion of macrophages, highlighting specificity of anti-Vsig4 Nbs for liver macrophage imaging. Ex vivo flow cytometry and immunohistochemistry analysis confirmed that anti-Vsig4 Nbs specifically targeted KCs but no other cell types in the liver. Upon induction of acute hepatitis using concanavalin A (ConA), down-regulation of the in vivo imaging signal obtained using anti-Vsig4 Nbs reflected reduction in KC numbers and transient modulation of Vsig4 expression on KCs. Overall, these results indicate that Nbs targeting Vsig4 as molecular imaging biomarker enable non-invasive monitoring of KCs during hepatic inflammation.

Novel microRNA-like viral small regulatory RNAs arising during human hepatitis A virus infection.

MicroRNAs (miRNAs), including host miRNAs and viral miRNAs, play vital roles in regulating host-virus interactions. DNA viruses encode miRNAs that regulate the viral life cycle. However, it is generally believed that cytoplasmic RNA viruses do not encode miRNAs, owing to inaccessible cellular miRNA processing machinery. Here, we provide a comprehensive genome-wide analysis and identification of miRNAs that were derived from hepatitis A virus (HAV; Hu/China/H2/1982), which is a typical cytoplasmic RNA virus. Using deep-sequencing and in silico approaches, we identified 2 novel virally encoded miRNAs, named hav-miR-1-5p and hav-miR-2-5p. Both of the novel virally encoded miRNAs were clearly detected in infected cells. Analysis of Dicer enzyme silencing demonstrated that HAV-derived miRNA biogenesis is Dicer dependent. Furthermore, we confirmed that HAV mature miRNAs were generated from viral miRNA precursors (pre-miRNAs) in host cells. Notably, naturally derived HAV miRNAs were biologically and functionally active and induced post-transcriptional gene silencing (PTGS). Genomic location analysis revealed novel miRNAs located in the coding region of the viral genome. Overall, our results show that HAV naturally generates functional miRNA-like small regulatory RNAs during infection. This is the first report of miRNAs derived from the coding region of genomic RNA of a cytoplasmic RNA virus. These observations demonstrate that a cytoplasmic RNA virus can naturally generate functional miRNAs, as DNA viruses do. These findings also contribute to improved understanding of host-RNA virus interactions mediated by RNA virus-derived miRNAs.

Conjugated bilirubin affects cytokine profiles in hepatitis A virus infection by modulating function of signal transducer and activator of transcription factors.

Hepatitis A virus (HAV) infection is the major cause of acute liver failure in paediatric patients. The clinical spectrum of infection is variable, and liver injury is determined by altered hepatic enzyme function and bilirubin concentration. We recently reported differences in cytokine profiles between distinct HAV-induced clinical courses, and bilirubin has been recognized as a potential immune-modulator. However, how bilirubin may affect cytokine profiles underlying the variability in the course of infection has not been determined. Herein, we used a transcription factor (TF) binding site identification approach to retrospectively analyse cytokine expression in HAV-infected children and to predict the entire set of TFs associated with the expression of specific cytokine profiles. The results suggested that modulation of the activity of signal transducers and activators of transcription proteins (STATs) may play a central role during HAV infection. This led us to compare the degree of STAT phosphorylation in peripheral blood lymphoid cells (PBLCs) from paediatric patients with distinct levels of conjugated bilirubin (CB). Low CB levels in sera were associated with increased STAT-1 and STAT-5 phosphorylation. A positive correlation was observed between the serum interleukin-6 (IL-6) content and CB values, whereas higher levels of CB correlated with reduced serum IL-8 values and with a reduction in the proportion of PBLCs positive for STAT-5 phosphorylation. When CB was used to stimulate patients' PBLCs in vitro, the levels of IL-6 and tumour necrosis factor-α were increased. The data showed that bilirubin plays a role in STAT function and affects cytokine profile expression during HAV infection.

[The hepatitis A virus: structural and functional organization of the genome, its molecular diagnostic value and cultivation].

The analysis of recently published data on hepatitis A virus (HAV) genome clinical features, molecular diagnostic value and cell culture propagation are reviewed. The growing need in the study of the genetic diversity of HAV isolates and the search of its possible new antigenic variants are underlined. The results of the cultivation of different HAV strains are analyzed for possible application in vaccine and diagnostic kit production.

Antibody-secreting cells with a phenotype of Ki-67low, CD138high, CD31high, and CD38high secrete nonspecific IgM during primary hepatitis A virus infection.

Although studies investigating the nature of Ab-secreting cells (ASCs) during acute infection with influenza or dengue virus found that the ASC response was dominated by virus-specific IgG secretion, the Ag specificity and phenotype of ASCs during primary acute viral infection were not identified. To this end, we investigated the nature of ASCs in direct ex vivo assays from patients with acute hepatitis A caused by primary infection with hepatitis A virus (HAV). We found that the frequency of CD27(high)CD38(high) ASCs was markedly increased in the peripheral blood during the acute phase of HAV infection. Moreover, substantial numbers of ASCs were non-HAV-specific and dominantly secreted IgM. We detected HAV-specific ASCs by staining with fluorochrome-tagged HAV-VP1 protein. As compared with HAV-specific ASCs, non-HAV-specific ASCs were Ki-67(low)CD138(high)CD31(high)CD38(high), demonstrating that non-HAV-specific ASCs had a bone marrow plasma cell-like phenotype whereas HAV-specific ASCs had a phenotype typical of circulating plasmablasts. These data suggest that non-HAV-specific ASCs might be mobilized plasma cells from the bone marrow or the spleen, whereas HAV-specific ASCs were newly generated plasmablasts. In this study, we provide evidence that pre-existing plasma cells are released into the circulation and contribute to Ag-nonspecific secretion of IgM during primary HAV infection.

Breastmilk hepatitis A virus RNA in nursing mothers with acute hepatitis A virus infection.

Breastmilk specimens from three women with acute hepatitis A virus (HAV) infection were studied. Anti-HAV immunoglobulin M and immunoglobulin G antibodies were detected in serum and breastmilk specimens of the three women. The three women also had serum HAV RNA. However, HAV RNA was detected only in two of the three breastmilk specimens. It is interesting that none of the three infants contracted clinical HAV infection. Furthermore, mothers with HAV infection should not be encouraged to discontinue breastfeeding.

Binding of hepatitis A virus to its cellular receptor 1 inhibits T-regulatory cell functions in humans.

BACKGROUND & AIMS: CD4+ T-regulatory (Treg) cells suppress immune responses and control self-tolerance and immunity to pathogens, cancer, and alloantigens. Most pathogens activate Treg cells to minimize immune-mediated tissue damage and prevent clearance, which promotes chronic infections. However, hepatitis A virus (HAV) temporarily inhibits Treg-cell functions. We investigated whether the interaction of HAV with its cellular receptor 1 (HAVCR1), a T-cell co-stimulatory molecule, inhibits the function of Treg cells to control HAV infection. METHODS: We studied the effects of HAV interaction with HAVCR1 on human T cells using binding, signal transduction, apoptosis, activation, suppression, cytokine production, and confocal microscopy analyses. Cytokines were analyzed in sera from 14 patients with HAV infection using bead arrays. RESULTS: Human Treg cells constitutively express HAVCR1. Binding of HAV to HAVCR1 blocked phosphorylation of Akt, prevented activation of the T-cell receptor, and inhibited function of Treg cells. At the peak viremia, patients with acute HAV infection had no Treg-cell suppression function, produced low levels of transforming growth factor-ß , which limited leukocyte recruitment and survival, and produced high levels of interleukin-22, which prevented liver damage. CONCLUSIONS: Interaction between HAV and its receptor HAVCR1 inhibits Treg-cell function, resulting in an immune imbalance that allows viral expansion with limited hepatocellular damage during early stages of infection-a characteristic of HAV pathogenesis. The mechanism by which HAV is cleared in the absence of Treg-cell function could be used as a model to develop anticancer therapies, modulate autoimmune and allergic responses, and prevent transplant rejection.