Evaluation of a new automated viral RNA extraction platform for hepatitis A virus and human norovirus in testing of berries, lettuce, and oysters.

Fruits, vegetables, and shellfish are often associated with outbreaks of illness caused particularly by human norovirus (HuNoV) and hepatitis A virus (HAV), the leading causative agents of foodborne illness worldwide. The aim of this study was to evaluate a new automated nucleic acid extraction platform (EGENE-UP EASYPREP) for enteric viruses in several at-risk food matrices and to test its limit of detection in comparison to a semi-automated method (EGENE-UP) using Boom methodology for nucleic acid extraction as suggested in the reference method ISO 15216-2:2019. Fresh and frozen raspberries, frozen blackberries, romaine lettuce and oyster digestive glands were artificially contaminated with HAV, HuNoV GII.4 or HuNoV GI.7 at 102, 103 or 104 genome copies/sample. Virus was then recovered from the food matrix using the ISO method. Viral RNA extracted from frozen berry samples by the automated system was purified on a column for additional removal of RT-qPCR inhibitors. For fresh raspberry, oysters, and romaine lettuce, the two extraction platforms were deemed equivalent. For frozen raspberry, the automated platform appeared to be more efficient for viral recovery, particularly for HAV and HuNoV GI at lower concentrations. With frozen blackberries, the two platforms may be considered equivalent for all targeted viruses. However, the automated method led to less sample-associated inhibition of the PCR, 56.5 % of samples versus 95.0 % for the semi-automated. We thus found that the automated extraction can be performed easily by users while obtaining equivalent or even superior results to the ISO 15216-2:2019 method, and therefore appears to be suitable for routine sanitary monitoring in food processing and for tracing outbreaks of illness.

Loxapine inhibits replication of hepatitis A virus in vitro and in vivo by targeting viral protein 2C.

No antiviral drugs currently are available for treatment of infection by hepatitis A virus (HAV), a causative agent of acute hepatitis, a potentially life-threatening disease. Chemical screening of a small-compound library using nanoluciferase-expressing HAV identified loxapine succinate, a selective dopamine receptor D2 antagonist, as a potent inhibitor of HAV propagation in vitro. Loxapine succinate did not inhibit viral entry nor internal ribosome entry site (IRES)-dependent translation, but exhibited strong inhibition of viral RNA replication. Blind passage of HAV in the presence of loxapine succinate resulted in the accumulation of viruses containing mutations in the 2C-encoding region, which contributed to resistance to loxapine succinate. Analysis of molecular dynamics simulations of the interaction between 2C and loxapine suggested that loxapine binds to the N-terminal region of 2C, and that resistant mutations impede these interactions. We further demonstrated that administration of loxapine succinate to HAV-infected Ifnar1-/- mice (which lack the type I interferon receptor) results in decreases in the levels of fecal HAV RNA and of intrahepatic HAV RNA at an early stage of infection. These findings suggest that HAV protein 2C is a potential target for antivirals, and provide novel insights into the development of drugs for the treatment of hepatitis A.

Concentration of foodborne viruses eluted from fresh and frozen produce: Applicability of ultrafiltration.

Foodborne illnesses involving raw and minimally processed foods are often caused by human noroviruses (HuNoV) and hepatitis A virus (HAV). Since food is contaminated usually with small numbers of virions, these must be eluted from the food surface and then concentrated for detection. The objective of this study was to optimize an ultrafiltration (UF) concentration method for HAV and HuNoVs present on various fresh and frozen produce. The detection range of the optimized method and its applicability to different food matrices was compared to the reference method ISO 15216-1:2017. Strawberry, raspberry, blackberry, lettuce, and green onion (25 g) were contaminated with HAV, HuNoV GI.7 and HuNoV GII.4 and then recovered therefrom by elution. A commercial benchtop UF device was used for the concentration step. Viral RNA was extracted and detected by RT-qPCR. From fresh strawberries, recovery of HAV loaded at 104 genome copies per sample was 30 ± 13 %, elution time had no significant impact, and UF membrane with an 80-100 kDa cut-off in combination with Tris-glycine elution buffer at pH 9.5 was found optimal. At lower copy numbers on fresh strawberry, at least 1 log lower numbers of HuNoV were detectable by the UF method (103 vs 104 GII.4 copies/sample and 101 vs 103 GI.7 copies/sample), while HAV was detected at 101 genome copies/sample by both methods. Except on raspberry, the UF method was usually equivalent to the ISO method regardless of the virus tested. The UF method makes rapid viral concentration possible, while supporting the filtration of large volume of sample. With fewer steps and shorter analysis time than the ISO method, this method could be suitable for routine analysis of viruses throughout the food production and surveillance chain.

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.

In-house RT-PCR detection of hepatitis A virus in human plasma pools used for the manufacture of medicinal products.

Hepatitis A virus (HAV) is the most frequent cause of viral hepatitis worldwide and is transmitted through the fecal-oral route. However, HAV can also be transmitted by blood-derived products. This is due to the fact that viremia occurs during the asymptomatic phase of HAV infection, enabling infected blood or plasma donations to occur. Viral inactivation/removal steps are included during manufacturing of plasmaderived products. However, HAV is a small non-enveloped virus very difficult to remove with traditional viral inactivation procedures. To accomplish European guidelines for pooled human plasma (treated for virus inactivation), plasma manufacturers have been implementing HAV nucleic acid test (NAT) screening on plasma pools. In this study, we validate an in-house multiplex reverse-transcription real-time PCR (RT-PCR) assay targeting HAV RNA and an internal control with hydrolysis probes for amplicon detection. The HAV RNA test was validated by assessing limit of detection, robustness, sensitivity and specificity according to European Pharmacopoeia (Ph. Eur.) guidelines. Our assay is able to detect 100 IU/mL of all human HAV genotypes that have been described so far. The multiplex assay shows remarkable sensitivity with a 95% lower limit of detection of 5.2 IU/mL. Also, our HAV test shows good robustness, precision, and specificity. We conclude that our assay broadly meets the requirements for its purpose. The implementation of this test in the production process of plasma-derived products will increase their safety.

Meta-analysis of the prevalence of norovirus and hepatitis a virus in berries.

Norovirus (NoV) and hepatitis A virus (HAV) stand as the predominant agents associated with viral foodborne infections. Outbreaks have been documented to be caused by various types of food items, including fresh and/or frozen berries. Comprehensive data concerning crucial viral pathogens in berries remain limited and are not currently available in aggregate form. Consequently, the present study aimed to compile the existing information regarding the prevalence of NoV and HAV in this matrix. Records of foodborne viruses were systematically extracted from database repositories up to December 2022, adhering to PRISMA standards and were subjected to a multilevel random effect meta-analysis model to determine the mean occurrence rate of NoV and HAV. A high heterogeneity across studies was observed (I2 = 82 %), reflecting variations in the prevalence of sampling locations, years, berry types, and sample conditions, among other potential contributing factors. The prevalence of NoV and HAV in berries was calculated at 2.12 % (95 % CI 1.74-2.59 %), and no statistically differences were observed among the viral types or genogroup categories. However, it is important to clarify that this estimate should be taken with caution given the high heterogeneity. There was no discernible correlation between viral prevalence and any particular berry type. However, there was a temporal correlation observed with the year of sampling, revealing a significantly decreasing trend throughout the study period. A significant influence of the sample condition (fresh or frozen) was observed in relation to the prevalence of NoV GII and HAV. Overall higher viral prevalences were identified in berries originating from African countries as compared to those sourced from other continents. It was also noted that the prevalence of NoV GI was significantly higher in samples collected directly from farms compared to those obtained from retailers. The outcomes of this comprehensive meta-analysis propose that while viral contamination of berries is diminishing in more recent times, the prevalence remains substantial in certain African countries, having a significant risk for foodborne infections. It is imperative to implement intervention strategies in these regions to enhance product safety.

Foodborne Viruses and Somatic Coliphages Occurrence in Fresh Produce at Retail from Northern Mexico.

Foodborne disease outbreaks linked to consumption of vegetables have been often attributed to human enteric viruses, such as Norovirus (NoV), Hepatitis A virus (HAV), and Rotavirus (RoV). Information about the occurrence of these viruses is scarce in many fresh-producing countries. Viral contamination detection of indicators, such as somatic coliphages, could indirectly reflect the presence of viral pathogens, being a valuable tool for better viral risk assessment in food industry. This study aimed to establish the occurrence and correlation of foodborne viruses and somatic coliphages in leafy greens in northern Mexico. A total of 320 vegetable samples were collected, resulting in 80 composite rinses, 40 of lettuce and 40 of parsley. Somatic coliphages were determined using the EPA 1602 method, while foodborne viruses (HAV, RoV, NoV GI, and GII) were determined by qPCR. The occurrence of RoV was 22.5% (9/40, mean 2.11 log gc/g) in lettuce and 20% (8/40, mean 1.91 log gc/g) in parsley. NoV and HAV were not detected in any samples. Somatic coliphages were present in all lettuce and parsley samples, with mean levels of 1.85 log PFU/100 ml and 2.28 log PFU/100 ml, respectively. Spearman analysis established the correlation of somatic coliphages and genomic copies of RoV, resulting in an r2 value of - 0.026 in lettuce and 0.349 in parsley. Although NoV or HAV were undetected in the samples, the presence of RoV is a matter of concern as leafy greens are usually eaten raw, which poses a potential risk of infection.

High-performance enrichment-based genome sequencing to support the investigation of hepatitis A virus outbreaks.

IMPORTANCE: This proof-of-concept study introduces a hybrid capture oligo panel for whole-genome sequencing of all six human pathogenic hepatitis A virus (HAV) subgenotypes, exhibiting a higher sensitivity than some conventional genotyping assays. The ability of hybrid capture to enrich multiple targets allows for a single, streamlined workflow, thus facilitating the potential harmonization of molecular surveillance of HAV with other enteric viruses. Even challenging sample matrices can be accommodated, making them suitable for broad implementation in clinical and public health laboratories. This innovative approach has significant implications for enhancing multijurisdictional outbreak investigations as well as our understanding of the global diversity and transmission dynamics of HAV.

Approaches to produce and characterize recombinant protein VP1-2A of HAV for serological rapid test application.

Studies reporting the expression of hepatitis A virus (HAV) structural proteins, specifically recombinant VP1-2A containing an immunogenic activity, use the Escherichia coli system. Recombinant HAV proteins may represent a source of less expensive antigens for application in different diagnostic platforms. However, the formation of insoluble aggregates is an obstacle to obtaining large amounts of HAV proteins in their native form. To overcome this obstacle, some approaches were applied in this study to improve purification, solubility, and protein expression levels. Critical properties were evaluated. The introduction of another insertion codon to increase the protein concentration and vector activity was observed and verified by SDS-PAGE. The expression was established with 0.4 mM IPTG for 4 h at 37 °C. The VP1 protein was partially soluble at an isoeletric point (pI) of 6.45. The majority of HAV VP1-2A proteins measured 45.19 kDa in size and had a homogeneity of 53.58%. Multi-antigen print immunoassay (MAPIA) showed antigenicity at different HAV VP1-2A concentrations, and microsphere-based immunoassays showed a specificity of 100% and a sensitivity of 84%. HAV VP1-2A was characterized using different sensitivity methods to prove its biological activity, indicating its use as a tool for the diagnosis of Hepatitis A virus infection.

Hepatitis A outbreak in a refugee shelter in Kiel, northern Germany.

In the spring of 2023, three Ukrainian war refugees from a municipal community shelter and a volunteer caregiver at an affiliated daycare center in Kiel, Germany, were diagnosed with infectious jaundice attributable to a single hepatitis A virus (HAV) subgenotype IA strain. Similar HAV sequences have been observed in Germany and other European countries for several years. One refugee and the volunteer required hospitalization. Four children were asymptomatically infected but excreted high levels of HAV ribonucleic acid in the stool. The infections were probably acquired in Germany, but a source could not be determined. The outbreak was contained through vaccination, increased hygiene, and education. The existing HAV vaccination recommendation for refugee shelter staff and volunteers should be consistently implemented.

Development of a cell-free toehold switch for hepatitis A virus type I on-site detection.

Picornavirus hepatitis A virus (HAV) is a common cause of hepatitis worldwide. It is spread primarily through contaminated food and water or person-to-person contact. HAV I has been identified as the most common type of human HAV infection. Here, we have developed a cell-free toehold switch sensor for HAV I detection. We screened 10 suitable toehold switch sequences using NUPACK software, and the VP1 gene was used as the target gene. The optimal toehold switch sequence was selected by in vivo expression. The best toehold switch concentration was further found to be 20 nM in a cell-free system. 5 nM trigger RNA activated the toehold switch to generate visible green fluorescence. The minimum detection concentration decreased to 1 pM once combined with NASBA. HAV I trigger RNA could be detected accurately with excellent specificity. In addition, the cell-free toehold switch sensor was verified in HAV I entities. The successful construction of the cell-free toehold switch sensor provided a convenient, rapid, and accurate method for HAV I on-site detection, especially in developing countries, without the involvement of expensive facilities and additional professional operators.

Divergent Genotype of Hepatitis A Virus in Alpacas, Bolivia, 2019.

Hepatitis A virus (HAV) is a common human pathogen found exclusively in primates. In a molecular and serologic study of 64 alpacas in Bolivia, we detected RNA of distinct HAV in ≈9% of animals and HAV antibodies in ≈64%. Complete-genome analysis suggests a long association of HAV with alpacas.

Detection of Hepatitis A RNA, Hepatitis E RNA, Human Adenovirus F DNA, and Norovirus RNA in Fresh and Frozen Berry Products at Point of Retail in Ireland.

Soft fruits are at particular risk of contamination with enteric viruses such as Hepatitis A virus (HAV), Hepatitis E Virus (HEV), Norovirus (NoV), Human Adenovirus (HAdV) and Sapovirus (SaV). The aim of this study was to investigate, for the first time, the presence of these biological agents in ready to eat (RTE) berries at point of retail in Ireland. A sampling strategy was designed in which RTE fresh and frozen strawberries and raspberries were purchased from five retailers between May and October 2018. Reverse Transcriptase Polymerase Chain Reaction (RT-qPCR) assays for HEV RNA, Nov RNA, SaV RNA, and human Adenovirus species F DNA (HAdV-F) were performed on 239 samples (25g portions). Viral nucleic acid was present in 6.7% (n = 16) of samples tested as follows: HAV RNA (n = 5), HAdV-F DNA (n = 5), HEV RNA (n = 3) and NoV GII RNA (n = 3). Sapovirus RNA was not detected in any product. No significant differences were found between berry type, fresh/frozen status, or supermarket source. This study suggests a risk that exists across all retail outlets however only low levels of nucleic acid ranging from 0 to 16 genome copies/g were present. Although these findings may reflect non-viable/non-infectious virus the continued provision of risk mitigation advice to consumers is warranted and further work is required to ensure control measures to reduce contamination are implemented and enforced.

Hepatitis A virus subgenotypes in Latvia, 2008-2021.

BACKGROUND: In Latvia outbreaks of the HAV were observed between 2008 and early 2010 and again in 2017-2018. However, the risks of introducing and spreading infection still exist, as the virus spreads easily when personal hygiene is not followed. METHODS: To determine the spread of HAV subgenotypes in the territory of Latvia the VP1/P2A genomic region of HAV was amplified and sequenced for 259 case serum samples. The study carried out a molecular biological investigation and molecular epidemiological investigation. Demographic data (sex, age), disease data (hepatitis symptoms, hospitalization, vaccination) and epidemiology data (part of the outbreak, possible source of infection, recent travel) were collected. Based on the obtained sequences, the phylogenetic tree was built and analyzed for the homology and belonging to different isolated HAV clusters from other countries. RESULTS: From the obtained data, it was concluded that HAV subgenotype IA had 13 clusters and 12 sporadic cases, HAV subgenotype IB had eight clusters and 11 sporadic cases, HAV subgenotype IIIA had one cluster and nine sporadic cases. It was found that the sources of infection among the investigated cases were different, they were mostly associated with contact with a patient with HAV, travel, as well as between persons who inject drugs and men who have sex with men, and the prevalence of HAV similar sequences was observed in different years. It was concluded that patients with HAV subgenotype IA had the longest hospitalization duration and averaged 9.3 days, while patients with subgenotype IB - 7.3 days, subgenotype IIIA - 7.7 days. Analyzing the data on vaccination, it was found that mostly all were not vaccinated or had an unknown vaccination status. CONCLUSIONS: All of this has led to the conclusion that the application of molecular biological methods of the HAV and a careful analysis of epidemiological data can help to better understand the ways of spreading the infection, investigate local outbreaks, detect cases of imported infection and track the recirculation of the virus.

Molecular diagnosis of patients with hepatitis A virus infection using amplicon-based nanopore sequencing.

High-throughput sequencing is a robust tool used for identifying and tracking pathogen outbreaks. Whole-genome sequencing of hepatitis A virus (HAV) remains poor due to ultra-low viral loads, limitations of next-generation sequencing technology, and its high costs in clinical applications. This study evaluated multiplex polymerase chain reaction (PCR)-based nanopore sequencing to obtain whole-genome sequences of HAV. The HAV genomes were obtained directly from patient specimens for a rapid molecular diagnosis of viral genotypes. Serum and stool samples were collected from six patients with hepatitis A infection. Amplicon-based nanopore sequencing was performed from the clinical specimens to identify HAV genotypes by acquiring nearly complete-genome sequences. TaqMan-based quantitative PCR (qPCR) was conducted to detect and quantify multiple HAV genes. Singleplex-based nanopore sequencing demonstrated high genome coverage rates (90.4-99.5%) of HAV within 8 h, at viral RNA loads of 10 to 105 copies/µL. TaqMan qPCR showed multiplex quantification of HAV genes namely, VP0, VP3, and 3C. This study provides useful insights into rapid molecular diagnosis during hepatitis A outbreaks and may ultimately augment public health disease surveillance in the hospital and epidemiology field.

Molecular epidemiology and characterization of endemic, epidemic and imported hepatitis A virus (HAV) strains in Hungary (2003-2022).

Hepatitis A virus (HAV) is one of the most important etiological agents of acute viral hepatitis but comprehensive molecular epidemiological study with chrono-phylogeographical data are not available from Hungary.Between 2003 and 2022, a total of 8,307 HAV infections were registered officially in Hungary of which 400 (4.8%) HAV IgM antibody-positive serum samples were collected countrywide. HAV genomic RNA was successfully detected in 216/400 (54%) sera by RT-PCR subsequently confirmed by sequencing. The complete nucleotide sequences of VP1 region were determined in 32 representative HAV strains. Based on the sequence analysis, 150 (69.4%) strains were characterized as HAV sub-genotype IA and 66 (30.6%) as sub-genotype IB, respectively. Based on the combined epidemiological and molecular data, epidemic, endemic, and imported HAV strains were also characterized. The first two registered countrywide outbreaks started among men-sex-with men (MSM) in 2011 (sub-genotype IA) and 2021 (sub-genotype IB), the continuously circulating endemic/domestic HAV strain (sub-genotype IA) in East Hungary and the travel-related sub-genotype IB strains from Egypt should be highlighted. All HAV strains are deposited in the HAVNET database (https://www.rivm.nl/en/havnet).In this 20-year-long comprehensive molecular epidemiological study, we report the genetic characterization and geographic distribution of endemic, epidemic and imported HAV strains for the first time in Hungary with continuous co-circulation of sub-genotypes IA and IB HAV strains since 2003. These data provide basic information about the HAV situation in the country in an international context and can promote more effective national public health intervention strategies for the prevention of HAV transmissions and infections.

Hepatitis A virus monitoring in wastewater: A complementary tool to clinical surveillance.

Monitoring wastewater is an effective tool for tracking information on trends of enteric viral dissemination. This study aimed to perform molecular detection and genetic characterization of HAV in wastewater and to correlate the results with those obtained from clinical surveillance. Wastewater samples (n=811) of the second most populous city in Argentina were collected from the main wastewater treatment plant (BG-WWTP, n=261), and at 7 local neighborhood collector sewers (LNCS, n=550) during 2017-2022. Clinical samples of acute hepatitis A cases (HA, n=54) were also analyzed. HAV molecular detection was performed by real time RT-PCR, and genetic characterization by RT-Nested PCR, sequencing and phylogenetic analysis. RNA-HAV was detected in sewage samples throughout the entire period studied, and detection frequencies varied according to the location and year (2.9% - 56.5%). In BG-WWTP, 23% of the samples were RNA-HAV+. The highest detection rates were in 2017 (30.0%), 2018 (41.7%) and 2022 (56.5%), which coincides with the highest number of HA cases reported. Twenty-eight (28) sequences were obtained (from clinical and sewage samples), and all were genotype IA. Two monophyletic clusters were identified: one that grouped clinical and wastewater samples from 2017-2018, and another with specimens from 2022, evidencing that environmental surveillance might constitute a replica of viral circulation in the population. These findings evidence that WBE, in a centralized and decentralized sewage monitoring, might be an effective strategy to track HAV circulation trends over time, contributing to the knowledge of HAV in the new post-vaccination epidemiological scenarios in Argentina and in Latin America.

Epidemiological characteristics of acute hepatitis A, 2013-2016: a cross-sectional study in Morocco.

BACKGROUND: Hepatitis A virus (HAV) is the common cause of acute hepatitis worldwide. Indeed, hepatitis A is endemic in developing countries such in Morocco and most residents are exposed in childhood. The characterisation of circulating strains of HAV remains crucial to understand the virological evolution and geo-temporal characteristics, which are essential for controlling infections and outbreaks. The purpose of the current study was the detection and characterisation of HAV strains circulating in Morocco by performing serological test, RT-PCR, sequencing and phylogenetic analysis. METHODS: In this cross-sectional study, 618 suspected acute hepatitis cases were examined by Architect HAV abIgM. Of the 162 positives, 64 underwent RNA extraction. None of the suspected cases was immune to HAV and none of them had received a blood transfusion. Samples found positive by RT-PCR using primers targeting the VP1/VP2A junction and the VP1/VP3 capsid region of HAV were subjected to sequencing and phylogenetic analyses. RESULTS: HAV Acute infection rate was 26.2% [95% CI, 22.8-29.9], while viraemia reached 45% (29/64) after amplification of the VP3/VP1 region. Phylogenetic analysis of the VP1/2A segment revealed the presence of sub-genotypes IA and IB. Eighty-seven percent of the strains belonged to the subgenotype IA, while twelve percent to IB subgenotype. CONCLUSION: This first molecular study of acute hepatitis A in Morocco provided information about genetic diversity of HAV, revealing the co-circulating of only two subgenotypes (IA and IB). Notably, subgenotype IA was found to be the predominant subgenotype in Morocco.

Masitinib Inhibits Hepatitis A Virus Replication.

The hepatitis A virus (HAV) infection causes acute hepatitis. HAV also induces acute liver failure or acute-on-chronic liver failure; however, no potent anti-HAV drugs are currently available in clinical situations. For anti-HAV drug screening, more convenient and useful models that mimic HAV replication are needed. In the present study, we established HuhT7-HAV/Luc cells, which are HuhT7 cells stably expressing the HAV HM175-18f genotype IB subgenomic replicon RNA harboring the firefly luciferase gene. This system was made by using a PiggyBac-based gene transfer system that introduces nonviral transposon DNA into mammalian cells. Then, we investigated whether 1134 US Food and Drug Administration (FDA)-approved drugs exhibited in vitro anti-HAV activity. We further demonstrated that treatment with tyrosine kinase inhibitor masitinib significantly reduced both HAV HM175-18f genotype IB replication and HAV HA11-1299 genotype IIIA replication. Masitinib also significantly inhibited HAV HM175 internal ribosomal entry-site (IRES) activity. In conclusion, HuhT7-HAV/Luc cells are adequate for anti-HAV drug screening, and masitinib may be useful for the treatment of severe HAV infection.