Viral hepatitis is a systemic infectious diseases caused by various hepatitis viruses, primarily leading to liver damage. It is widely prevalent worldwide, with hepatitis viruses categorized into five types: hepatitis A, B, C, D, and E, based on their etiology. Currently, the detection of hepatitis viruses relies on methods such as enzyme-linked immunosorbent assay (ELISA), immunoelectron microscopy to observe and identify viral particles, and in situ hybridization to detect viral DNA in tissues. However, these methods have limitations, including low sensitivity, high error rates in results, and potential false negative reactions due to occult serum infection conditions. To address these challenges, we have designed an AuNPs-DNA walker method that uses gold nanoparticles (AuNPs) and complementary DNA strands for detecting viral DNA fragments through a colorimetric assay and fluorescence detection. The DNA walker, attached to gold nanoparticles, comprises a long walking strand with a probe sequence bound and stem-loop structural strands featuring a modified fluorescent molecule at the 3' end, which contains the DNAzyme structural domain. Upon the addition of virus fragments, the target sequence binds to the probe chains. Subsequently, the long walking strand is released and continuously hybridizes with the stem-loop structural strand. The DNAzyme undergoes hydrolytical cleavage by Mg2+, breaking the stem-loop structural strand into linear single strands. As a result of these structural changes, the negative charge density in the solution decreases, weakening spatial repulsion and rapidly reducing the stability of the DNA walker. This leads to aggregation upon the addition of a high-salt solution, accompanied by a color change. Virus typing can be performed through fluorescence detection. The innovative method can detect DNA/RNA fragments with high specificity for the target sequence, reaching concentrations as low as 1 nM. Overall, our approach offers a more convenient and reliable method for the detection of hepatitis viruses.

In 2018, there was a hepatitis A outbreak in Japan, and hepatitis A virus (HAV) infection is considered a sexually transmitted disease. In general, patients with hepatitis A should be given attention, and this disease should be prevented more than ever. The Japan Agency for Medical Research and Development (AMED) Hepatitis A and E viruses (HAV and HEV) Study Group has worked on the project to create "Recent Advances in Hepatitis A Virus (HAV) Research and Clinical Practice Guidelines for HAV Infection in Japan". The group consists of expert hepatologists and virologists who gathered at virtual meeting on August 5, 2023. Data about the pathogenesis, infection routes, diagnosis, complications, several factors for the severities, vaccination, and current and future treatments for hepatitis A were discussed and debated for a draft version. The participants assessed the quality of cited studies. The finalized recommendations are presented in this review. The recent advances in HAV research and clinical practice for HAV infection in Japan, have been reviewed by the AMED HAV and HEV Study Group.

Hepatitis A virus (HAV) is a common infection that is transmitted through the fecal-oral route, shed in the stool of infected individuals, and spread either by direct contact or by ingesting contaminated food or water. Each year, approximately 1.4 million acute cases are reported globally with a major risk factor for exposure being low household socioeconomic status. Recent trends show a decrease in anti-HAV antibodies in the general population, with concomitant increases in the numbers of HAV outbreaks. In line with a recreational water study, this effort aims to assess the prevalence of salivary IgG antibodies against HAV and subsequent incident infections (or immunoconversions) in visitors to a tropical beach impacted by a publicly owned treatment works (POTW). We applied a multiplex immunoassay to serially collected saliva samples gathered from study participants who recreated at Boquerón Beach, Puerto Rico. Analysis of assay results revealed an immunoprevalence rate of 16.17% for HAV with 1.43% of the cohort immunoconverting to HAV. Among those who immunoconverted, 10% reported chronic gastrointestinal symptoms and none experienced diarrhea. Tests on water samples indicated good water quality with low levels of fecal indicator bacteria; however, the collection and analysis of saliva samples afforded the ability to detect HAV infections in beachgoers. This rapid assay serves as a cost-effective tool for examining exposure to environmental pathogens and can provide critical information to policy makers, water quality experts, and risk assessment professionals seeking to improve and protect recreational water and public health.

Hepatitis A virus (HAV) is a causative agent of acute viral hepatitis for which an effective vaccine has been developed. Here we describe ultra-deep pyrosequences (UDPSs) of HAV 5'-untranslated region (5'UTR) among cases of the same outbreak, which arose from a single source, associated with a revolving sushi bar. We determined the reference sequence from HAV-derived clone from an attendant by the Sanger method. Sixteen UDPSs from this outbreak and one from another sporadic case were compared with this reference. Nucleotide errors yielded a UDPS error rate of < 1%. This study confirmed that nucleotide substitutions of this region are transition mutations in outbreak cases, that insertion was observed only in non-severe cases, and that these nucleotide substitutions were different from those of the sporadic case. Analysis of UDPSs detected low-prevalence HAV variations in 5'UTR, but no specific mutations associated with severity in these outbreak cases. To our surprise, HAV strains in this outbreak conserved HAV IRES sequence even if we performed analysis of UDPSs. UDPS analysis of HAV 5'UTR gave us no association between the disease severity of hepatitis A and HAV 5'UTR substitutions. It might be more interesting to perform ultra-deep sequencing of full length HAV genome in order to reveal possible unknown genomic determinants associated with disease severity. Further studies will be needed.

A correlation between hepatitis A virus (HAV) genomes and the clinical severity of hepatitis A has not been established. The viral load in sera of hepatitis A patients was examined to determine the possible association between hepatitis A severity and HAV replication. One hundred sixty-four serum samples from 91 Japanese patients with sporadic hepatitis A, comprising 11 patients with fulminant hepatitis, 10 with severe acute hepatitis, and 70 with self-limited acute hepatitis, were tested for HAV RNA. The sera included 83 serial samples from 20 patients. Viral load was measured by real-time RT-PCR. The detection rates of HAV RNA from fulminant, severe acute, and acute hepatitis were 10/11 (91%), 10/10 (100%), and 55/70 (79%), respectively. Mean values of HAV RNA at admission were 3.48 ± 1.30 logcopies/ml in fulminant, 4.19 ± 1.03 in severe acute, and 2.65 ± 1.64 in acute hepatitis. Patients with severe infection such as fulminant hepatitis and severe acute hepatitis had higher initial viral load than patients with less severe infection (P < 0.001). Viremia persisted for 14.2 ± 5.8 days in patients with severe infection and 21.4 ± 10.6 days in those with acute hepatitis after clinical onset (P = 0.19). HAV RNA was detectable quantitatively in the majority of the sera of hepatitis A cases during the early convalescent phase by real-time PCR. Higher initial viral replication was found in severely infected patients. An excessive host immune response might follow, reducing the viral load rapidly as a result of the destruction of large numbers of HAV-infected hepatocytes, and in turn severe disease might be induced.

Mutations in the internal ribosome entry site (IRES) of hepatitis A virus (HAV) have been associated with enhanced in vitro replication and viral attenuation in animal models. To address the possible role of IRES variability in clinical presentation, IRES sequences were obtained from HAV isolates associated with benign (n = 8) or severe (n = 4) hepatitis. IRES activity was assessed using a bicistronic dual-luciferase expression system in adenocarcinoma (HeLa) and hepatoma (HuH7) cell lines. Activity was higher in HuH7 than in HeLa cells, except for an infrequently isolated genotype IIA strain. Though globally low, significant variation in IRES-dependent translation efficiency was observed between field isolates, reflecting the low but significant genetic variability of this region (94.2% +/- 0.5% nucleotide identity). No mutation was exclusive of benign or severe hepatitis, and variations in IRES activity were not associated with a clinical phenotype, indirectly supporting the preponderance of host factors in determining the clinical presentation.

We analysed the association of the 5' nontranslated region (5'NTR), nonstructural proteins 2B and 2C of the hepatitis A virus (HAV) genome, whose mutations have previously been shown to be important for enhanced replication in cell culture systems, in order to align all our data and examine whether genomic differences in HAV are responsible for the range of clinical severities.

Our accumulated HAV strains of 5'NTR [nucleotide(nt) 200 and 500], entire 2B and 2C from 25 Japanese patients with sporadic hepatitis A, consisting of seven patients with fulminant hepatitis (FH), five with severe acute hepatitis (AHs) and 13 with self-limited acute hepatitis (AH), in whom the sequences of all three regions were available, were subjected to phylogenetic analysis.

Fulminant hepatitis patients had fewer nucleotide substitutions in 5'NTR, had a tendency to have more amino acid (aa) substitutions in 2B and had fewer aa substitutions in 2C than AH patients. Four FH and two AHs with a higher viral replication were located in the near parts of the phylogenetic trees, indicating the association between the severity of hepatitis A and genomic variations in 5'NTR, 2B and 2C of HAV.

Our study suggests that genetic variations in HAV not in one specific region but in 5'NTR, 2B and 2C might cooperatively influence replication of the virus, and thereby affect virulence. Viral factors should be considered and examined when discussing the mechanisms responsible for the severity of hepatitis A.

In our recent study of the full-length hepatitis A virus (HAV) genome from some patients with fulminant hepatitis and acute hepatitis, possible associations were suggested between the severity of hepatitis A and the amino acid substitutions in the nonstructural protein 2B. We therefore analyzed HAV 2B from many patients with various clinical disease severities.

Serum samples from 30 Japanese patients with sporadic hepatitis A from five widely separated regions of Japan, comprising nine patients with fulminant hepatitis (FH), six with severe acute hepatitis (AHs), and 15 with acute hepatitis (AH), were examined for HAV RNA. The entire sequences of HAV 2B were analyzed.

Compared with the sequence of the wild-type HAV strain GBM, nucleotide sequences of 2B had homology of 94.5 +/- 1.0% in FH, 95.2 +/- 1.2% in AHs, and 95.1 +/- 1.8% in AH. Deduced amino acid sequences had homology of 97.5 +/- 2.1% in FH, 97.9 +/- 2.4% in AHs, and 98.5 +/- 1.3% in AH. Differences were not statistically significant among the three groups. The average number of amino acid mutations between amino acids 100 and 200 was 5.0 +/- 5.2 per case in FH, 4.0 +/- 6.0 in AHs, and 1.9 +/- 2.9 in AH. The differences between FH and AH, AHs and AH, and between severe cases (FH and AHs) and nonsevere cases (AH) were not statistically significant (P = 0.13, P = 0.45, and P = 0.10, respectively).

There were no obvious differences in the sequences among FH, AHs, and AH throughout the 2B region, but there seemed to be more mutations in the strains obtained from FH and AHs patients than in those obtained from AH patients in the central part of HAV 2B.

The Northeast region is the location of most cases of acute hepatitis A virus (HAV) in Brazil. In the present study, the genotypes of HAV strains from Pernambuco State, one of most populous states in the Northeast region, were characterized. Blood samples positive for anti-HAV IgM from 145 individuals (mean age = 29.1 years), collected during 2002 and 2003, were submitted to nested RT-PCR for amplification of the 5'non-translated region (5'NTR) and VP1/2A regions of the HAV genome. The VP1/2A and 5'NTR regions were amplified in 39 and 21% of the samples, respectively. Nucleotide sequencing was carried out in 46% of VP1/2A and in 53% of 5'NTR isolates. The identity in nucleotide sequence of the VP1/2A region ranged from 93.6 to 100.0%. Phylogenetic analysis of the VP1/2A sequences showed that 65% belong to sub-genotype IA and 35% to sub-genotype IB. Co-circulation of both sub-genotypes was observed in the two years studied. Distinct clusters of highly related sequences were observed in both sub-genotypes, suggesting endemic circulation of HAV strains in this area. In the 5'NTR isolates, 92.7-99.2% identity was observed and two isolates presented one deletion at position 413. Phylogenetic analysis showed that genotype IA strains cluster in the tree in the same way as genotype IB strains, but one IIIA isolate from Spain clusters with genotype IB strains. These results do not allow us to state that 5'NTR could be used to genotype HAV sequences. This is the first report of co-circulation of sub-genotypes IA and IB in this region, providing additional information about the molecular epidemiology of HAV strains in Brazil.

Current serologic tests provide the foundation for diagnosis of hepatitis A and hepatitis A virus (HAV) infection. Recent advances in methods to identify and characterize nucleic acid markers of viral infections have provided the foundation for the field of molecular epidemiology and increased our knowledge of the molecular biology and epidemiology of HAV. Although HAV is primarily shed in feces, there is a strong viremic phase during infection which has allowed easy access to virus isolates and the use of molecular markers to determine their genetic relatedness. Molecular epidemiologic studies have provided new information on the types and extent of HAV infection and transmission in the United States. In addition, these new diagnostic methods have provided tools for the rapid detection of food-borne HAV transmission and identification of the potential source of the food contamination.

The purpose of the present study was to investigate the influence of viral load on disease severity and analyze the possible relationship of the load of hepatitis A virus (HAV) with disease severity and laboratory findings.

Fifty-eight patients diagnosed with acute hepatitis A were used in the current study, of whom 12 patients progressed to severe acute hepatitis (s-AH) defined on the basis of a prothrombin time (PT) of <40% and 46 patients were diagnosed as having mild acute hepatitis (m-AH). The load of HAV was measured with real-time polymerase chain reaction.

Peak viral load showed a significant correlation with alanine aminotransferase (ALT) (r = 0.363, P = 0.0048) and PT levels (r = -0.330, P = 0.0110). In terms of disease severity, there was a significant correlation with ALT (r = 0.462, P = 0.0012) and PT levels (r = 0.400, P = 0.0059) in the m-AH group, but not in the s-AH group. A significant positive correlation of peak viral load with the C-reactive protein level (r = 0.270, P = 0.0400) and a significant negative correlation of peak viral load with the platelet count (r = -0.313, P = 0.0015) was also found.

The load of HAV was closely correlated with liver damage and disease severity in m-AH, but not in s-AH. The load of HAV was also closely associated with the increase in C-reactive protein level and enhancement of thrombocytopenia.

Investigation of hepatitis A virus (HAV) outbreaks often implies nucleotide sequence analysis. As an alternative method for the identification of related strains, single strand conformation polymorphism method (SSCP) was compared to sequence analysis. Twenty-three strains from sporadic and outbreak cases were studied retrospectively. SSCP, sequence identity and phylogenetic analyses were conducted on a 267 bp fragment of the VP1-2A variable region. The results of SSCP pattern comparison and sequence identity were highly correlated (r = 0.92, P < 0.001). If SSCP showed similar patterns, the VP1-2A fragments had a high and significant probability to have a sequence identity over 99.6%. Results were concordant for outbreak strains. The only discordant result concerned a cluster of three sporadic cases evidenced by phylogenetic analysis while SSCP showed similar patterns for only two of these three cases. A prospective SSCP analysis of a recent HAV outbreak confirmed the reliability of this technique. SSCP may thus provide a rapid and cost-effective tool for preliminary investigation of HAV outbreaks, before undertaking exhaustive nucleotide sequence analysis.

Fulminant hepatitis is a severe complication of hepatitis A virus infection. Its mechanism is unknown. Liver transplantation can be necessary, but spontaneous recovery is frequent. There are no data on the level of viral replication according to the clinical form of hepatitis A. We reviewed the files of 50 patients with acute hepatitis A. Nineteen patients had fulminant hepatitis (defined by encephalopathy and factor V <50%), and, from them, 10 patients underwent transplantation. Hepatitis A virus (HAV) RNA was quantified by real-time PCR on sera obtained at admission. The genotype was determined by phylogenetic analysis of HAV RNA. HAV RNA was detected in serum by RT-PCR in 39 out of 50 patients. Encephalopathy and low factor V level were significantly related to female gender, HAV PCR negativity (9/19 vs. 5/31, respectively; P =.03), a low serum HAV RNA level (log, 3.6 +/- 0.6 vs. 4.4 +/- 0.9, respectively; P =.02), genotypes other than IA, and acetaminophen intake. In multivariate analysis, low or undetectable HAV viral load and a high bilirubin level were independently associated with both low factor V levels and fulminant hepatitis and also with death or transplantation. In conclusion, HAV-related liver failure is due to an excessive host response associated with a marked reduction in viral load. Serum HAV RNA assay could be of help in the management of severe hepatitis A.

The correlation between the length of viremia as detected by reverse transcriptase-polymerase chain reaction (RT-PCR) and the clinical course of hepatitis A virus (HAV) infection was studied. Sixty-six consecutive patients with acute hepatitis A who were admitted to hospital in two infectious disease units in southern Italy were enrolled: 57 had a self-limited course of the disease (typical course), 4 a prolonged course, and 5 relapsing hepatitis. Plasma HAV RNA was sought by RT-PCR, using primers made at 5'-NTR of HAV, designed to amplify a 273-bp fragment and detected by 2% agarose gel and by hybridization with a specific biotinylated probe. In four patients with prolonged acute hepatitis A, the plasma HAV RNA, which was positive on the day of admission to hospital, was found to be negative from day 62, 46, 84, and 105, respectively, after the onset of the symptoms. In patients with relapsing hepatitis, HAV viremia paralleled the clinical and biochemical course of disease. In all patients with a typical self-limiting course, clearance of plasma HAV RNA was observed within 20 days of the onset of symptoms. In most patients, plasma HAV viremia became undetectable before the normalization of serum aminotransferases, underlining the importance of the immune reaction in the pathogenesis of acute hepatitis A. The data also suggest that the detection of plasma HAV RNA after 20 days of illness may predict a prolonged course of the disease, but relapsing hepatitis remains unpredictable on the basis of plasma HAV determination.

Hepatitis A virus (HAV) infection is a major public health problem worldwide. The infection does not induce any visible cytopathic effects or interfere with macromolecular synthesis in host cells. However, the hepatitis B and C viruses have recently been reported to activate intracellular signals. To clarify the effects of HAV infection on intracellular signalling, we examined the influence of 9 FLAG-tagged HAV proteins (VP2, VP3, VP1-2A, 2B, 2C, 3A, 3BC, 3C and 3D) on signal transduction pathways.

Viral protein expression vectors were co-transfected into HeLa cells with reporter plasmids controlled by a synthetic promoter containing direct repeats of the cyclic AMP response element (CRE), serum response factor (SRF), activator protein 1 (AP-1), nuclear factor kappaB (NF-kappaB) or serum response element (SRE). Cells were harvested 42 h after transfection and luciferase assays were performed. Viral protein activation twice that of the control was defined as significant.

VP3 induced an SRE-associated signal 2.2 +/- 0.3 times higher than that of control. VP3 did not activate CRE-, SRF-, AP-1- or NF-kappaB- associated signalling. The other HAV proteins tested also failed to induce these pathways.

HAV interacts with the host signalling mechanism, and HAV VP3, different from HBX and hepatitis C core protein, may activate only SRE-associated intracellular signalling, a pathway associated with cell proliferation and differentiation.

Duck hepatitis B virus (DHBV) belongs to the Hepadnaviridae family, which includes human Hepatitis B virus (HBV) and Woodchuck hepatitis virus. It is widely distributed in wild and domestic ducks due to congenital transmission. HBV is a worldwide health problem, with carriers at risk of developing cirrhosis and liver cancer. Medical staff and scientists working with HBV must be vaccinated because of its contagious nature. DHBV is a safe surrogate for HBV because of their similarities. Collection of serum and blood samples on filter paper has been used to screen for metabolic disorders, genetic diseases, and viral infection and for evolutionary studies of the genome. In this study, DHBV from serum and blood dried on filters was detected by PCR. A 0.1-microl sample was sufficient for detection. The immobilization potential of filter papers for DHBV was examined, and the highest yield of PCR products was observed with Whatman paper. Dried serum was stable under different storage temperatures for 4 weeks, but the yields of PCR products decreased when the temperature was >or=4 degrees C. The optimal condition for storage was -70 degrees C. A newly developed quantitative PCR based on monitoring the amplification by measuring the increase in fluorescence caused by the binding of SYBR green dye to double-stranded products was applied herein. DHBV genomic DNA cloned in a plasmid was used for the generation of standard DHBV DNA for quantitative PCR. It validated results from PCR in terms of the copy number of DHBV particles. The specificity of PCR was demonstrated by melting curve analysis, and the differentiation of two DHBV isolates amplified from dried serum was demonstrated based on their melting temperatures determined by GC contents and sequence. It was easier and simpler than other PCR-based DNA techniques. The use of serum dried on filters allows samples from distant field for which cold storage and transportation are a problem to be mailed to the diagnostic laboratory. Samples can be archived for comparison and used as a source of DNA for cloning and sequencing.

Type A hepatitis still poses a considerable problem worldwide. Why some patients progress to fulminant type A hepatitis and others do not is still unknown. To examine whether genomic differences of hepatitis A virus (HAV) are responsible for the severity of the disease, we analyzed the whole HAV genomes from patients with fulminant and self-limited acute type A hepatitis.

Sera from three patients with sporadic type A fulminant hepatitis (FH) and three patients with acute hepatitis (AH) were examined for HAV RNA. Full-length nucleotide sequences were determined using long reverse transcription polymerase chain reaction, 5' and 3' rapid amplification of cDNA ends methods, and direct sequencing. The amino acid sequences were deduced from the nucleotide sequences.

HAV RNA was detected in all six patients examined. From the sequence of viral protein 1/2A, all cases were revealed to be genotype IA. By comparing with genotype IA, wild-type HAV strain GBM, the analysis of whole genomes from the six cases showed no specific substitutions between FH and AH. Completely identical nucleotide sequences were observed at 3' non-translated region (NTR) in all six cases. In 5'NTR, less nucleotide substitutions were found in FH than in AH, and in the non-structural protein 2B region, a little more amino acid substitutions seemed to be found in FH than in AH.

This study showed that full-length HAV could be analyzed from serum samples. Although there were no unique nucleotide or amino acid substitutions, possible associations were suggested between the severity of type A hepatitis and the nucleotide substitutions in 5'NTR and the amino acid substitutions in 2B.