Ongoing outbreak of hepatitis A associated with sexual transmission among men who have sex with men, Portugal, October 2023 to April 2024.

An outbreak of hepatitis A is ongoing in Portugal, with 71 confirmed cases from 7 October 2023 to 24 April 2024. Most cases are male, aged 18-44 years, with many identifying as men who have sex with men (MSM) and reported as suspected sexual transmission. Phylogenetic analysis identified the subgenotype IA, VRD 521-2016 strain, last observed in an MSM-associated multi-country outbreak in 2016 to 2018. We wish to alert colleagues in other countries to investigate potential similar spread.

A Reliable Multifaceted Solution against Foodborne Viral Infections: The Case of RiLK1 Decapeptide.

Food-borne transmission is a recognized route for many viruses associated with gastrointestinal, hepatic, or neurological diseases. Therefore, it is essential to identify new bioactive compounds with broad-spectrum antiviral activity to exploit innovative solutions against these hazards. Recently, antimicrobial peptides (AMPs) have been recognized as promising antiviral agents. Indeed, while the antibacterial and antifungal effects of these molecules have been widely reported, their use as potential antiviral agents has not yet been fully investigated. Herein, the antiviral activity of previously identified or newly designed AMPs was evaluated against the non-enveloped RNA viruses, hepatitis A virus (HAV) and murine norovirus (MNV), a surrogate for human norovirus. Moreover, specific assays were performed to recognize at which stage of the viral infection cycle the peptides could function. The results showed that almost all peptides displayed virucidal effects, with about 90% of infectivity reduction in HAV or MNV. However, the decapeptide RiLK1 demonstrated, together with its antibacterial and antifungal properties, a notable reduction in viral infection for both HAV and MNV, possibly through direct interaction with viral particles causing their damage or hindering the recognition of cellular receptors. Hence, RiLK1 could represent a versatile antimicrobial agent effective against various foodborne pathogens including viruses, bacteria, and fungi.

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.

Factors associated with hepatitis A susceptibility among men who have sex with men using HIV pre-exposure prophylaxis in Northeastern Brazil: A cross-sectional study.

Hepatitis A virus (HAV) infection has disproportionately affected more men who have sex with men (MSM), occurring in outbreaks, despite being vaccine-preventable. We determined the prevalence and factors associated with HAV susceptibility among cisgender MSM on HIV pre-exposure prophylaxis (PrEP) in Northeastern Brazil. From September 30, 2021 to June 19, 2023, 282 cisgender MSM receiving HIV PrEP were enrolled into this cross-sectional study. Sociodemographic and clinical information were collected. Blood samples were collected for screening of sexually transmitted infections (STIs) and serum samples were tested for IgM and total anti-HAV antibodies. Non-reactive results for total anti-HAV antibodies were found in 106 of 282 (37.6%) participants. Factors associated with HAV susceptibility included age <30 years (prevalence ratio [PR]: 2.02; 95% confidence interval [95% CI]: 1.61-2.53), having health insurance (PR: 1.39; 95% CI: 1.19-1.64), sex only with cisgender men (PR: 1.52; 95% CI: 1.23-1.89), non-steady partner (PR: 1.20; 95% CI: 1.01-1.43) and no lifetime history of STIs (PR: 1.25; 95% CI: 1.03-1.53). Identifying clinical correlates of HAV susceptibility in key populations is a fundamental step towards development of public policy focused on prevention, especially following the recent hepatitis A outbreak in Brazil.

Assessing Immunity to Hepatitis A and Hepatitis B Among Individuals in Custody Living With HIV.

Screening upon entry into prison for hepatitis A virus (HAV) and hepatitis B virus (HBV) provides an ideal public health opportunity to offer vaccination to individuals who are nonimmune. We conducted a retrospective review of HAV and HBV immunity among adults living with HIV in the Illinois Department of Corrections between January 1, 2019, and December 31, 2019. The primary objective was to assess rates of HAV and/or HBV immunity in individuals with HIV. In total, 436 people were included in the study. Of 425 patients who had data for HAV vaccination, 335 were immune. Of 421 patients who had data for HBV vaccination, 272 were immune. Of the 149 patients who were nonimmune to HBV, 22 had active HBV and 6 had an equivocal HBV surface antibody and negative HBV surface antigen. In total, 212 (52%) were immune to both HAV and HBV, and 31 (8%) had no immunity to either HAV or HBV. These data demonstrate an important opportunity to discuss and provide vaccination while in custody.

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.

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.

Trends of Hepatitis A Virus Infection in Poland: Assessing the Potential Impact of the COVID-19 Pandemic and War in Ukraine.

Hepatitis A virus (HAV) is the most common cause of acute viral hepatitis, which is preventable by vaccination. This study analyzed trends of HAV infections in Poland according to socio-demographic features in the years 2009-2022 and assessed the potential impact of the COVID-19 pandemic (2020-2023) and the migration of war refugees from Ukraine (since February 2022). In 2009-2022, 7115 new cases of HAV infection were diagnosed in Poland, especially among men (66.4%) and in urban areas (77.4%). Infections among men were most common at the age of 25-34 (median rate 0.43 per 105) and in women aged 15-24 (median rate 0.39 per 105). Analysis of the 14-year frequency of HAV infections exhibited three trends, regardless of gender, age, and residence. The infections revealed a downward trend in 2009-2014, increased significantly in 2014-2018, and decreased again after 2018. A particularly rapid increase in HAV infections occurred between March 2017 and February 2018 (median rate 0.79 per 105). The high level of new infections persisted until the beginning of the COVID-19 pandemic, at which point it dropped significantly but did not reach the level recorded before March 2017. During the Omicron SARS-CoV-2 dominance period, the median rate of HAV infections was 0.053 per 105, with a four-fold increase being observed from February 2022 (when the migration of war refugees from Ukraine began) to August 2022. The presented results can serve as a reference point for further observations in Central Europe. The HAV epidemiological situation is unlikely to escalate in Poland but requires further monitoring.

"PROTAC" modified dihydroquinolizinones (DHQs) that cause degradation of PAPD-5 and inhibition of hepatitis A virus and hepatitis B virus, in vitro.

Dihydroquinolizinones (DHQs) that inhibit cellular polyadenylating polymerases 5 and 7 (PAPD5 & 7), such as RG7834, have been shown to inhibit both hepatitis A (HAV) and hepatitis B virus (HBV) in vitro and in vivo. In this report, we describe RG7834-based proteolysis-targeting chimeras (PROTACs), such as compound 12b, (6S)-9-((1-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)-21-oxo-3,6,9,12,15,18-hexaoxa-22-azapentacosan-25-yl)oxy)-6-isopropyl-10-methoxy-2-oxo-6,7-dihydro-2H-pyrido[2,1-a]isoquinoline-3-carboxylic acid. The PROTAC DHQs described here inhibited an HAV reporter virus in vitro with an IC50 of 277 nM. Although the PROTAC DHQs were also inhibitory to HBV, their activities were substantially less potent against HBV in vitro, being in the 10 to 20 µM range, based on the reduction of HBsAg and HBV mRNA levels. Importantly, unlike RG7834, the incubation of cells in vitro with PROTAC DHQ 12b resulted in the degradation of PAPD5, as expected for a PROTAC compound, but curiously not PAPD7. PAPD5 polypeptide degradation was prevented when a proteasome inhibitor, epoxomicin, was used, indicating that proteasome mediated proteolysis was associated with the observed activities of 12b. Taken together, these data show that 12b is the first example of a PROTAC that suppresses both HAV and HBV that is based on a small molecule warhead. The possibility that it has mechanisms that differ from its parent compound, RG7834, and has clinical value, is discussed.

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.

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.

Heat stability of foodborne viruses - Findings, methodological challenges and current developments.

Heat treatment of food represents an important measure to prevent pathogen transmission. Thus far, evaluation of heat treatment processes is mainly based on data from bacteria. However, foodborne viruses have gained increasing attention during the last decades. Here, the published literature on heat stability and inactivation of human norovirus (NoV), hepatitis A virus (HAV) and hepatitis E virus (HEV) was reviewed. Data for surrogate viruses were not included. As stability assessment for foodborne viruses is often hampered by missing infectivity assays, an overview of applied methods is also presented. For NoV, molecular capsid integrity assays were mainly applied, but data from initial studies utilizing novel intestinal enteroid or zebrafish larvae assays are available now. However, these methods are still limited in applicability and sensitivity. For HAV, sufficient cell culture-based inactivation data are available, but almost exclusively for one single strain, thus limiting interpretation of the data for the wide range of field strains. For HEV, data are now available from studies using pig inoculation or cell culture. The results of the reviewed studies generally indicate that NoV, HAV and HEV possess a high heat stability. Heating at 70-72 °C for 2 min significantly reduces infectious titers, but often does not result in a >4 log10 decrease. However, heat stability greatly varied dependent on virus strain, matrix and heating regime. In addition, the applied method largely influenced the result, e.g. capsid integrity assays tend to result in higher measured stabilities than cell culture approaches. It can be concluded that the investigated foodborne viruses show a high heat stability, but can be inactivated by application of appropriate heating protocols. For HAV, suggestions for safe time/temperature combinations for specific foods can be derived from the published studies, with the limitation that they are mostly based on one strain only. Although significant improvement of infectivity assays for NoV and HEV have been made during the last years, further method development regarding sensitivity, robustness and broader applicability is important to generate more reliable heat inactivation data for these foodborne viruses in future.

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.

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.

Prevalence of viral hepatitis (A, B, C, and E) infection and co-infection among hospitalized children in Cairo, Egypt.

Viral hepatitis is considered a public health issue facing the entire world. The World Health Organization encouraged all countries to work together to eliminate this fatal infection and achieve the 2030 agenda. The present study aimed to investigate the silent infection of viral hepatitis (A, B, C, and E) among hospitalized children in Cairo, Egypt, to control and avoid chronic infection early on. This cross-sectional study included 184 randomly selected hospitalized children from three different hospitals in Cairo, Egypt. They were children aged between a few months to 15 years to determine viral hepatitis infection and co-infection. Antibodies to hepatitis A virus (HAV IgM), hepatitis E virus (HEV IgM), hepatitis C virus (HCV Ab), and hepatitis B virus surface antigen (HBs Ag) were performed by ELISA. If the ELISA results were positive, the viral load was quantified by real-time polymerase chain reaction (RT-PCR). Other laboratory investigations included alanine aminotransferase, aspartate aminotransferase, albumin, and complete blood count. Only five children (2.71%) had HCV Ab positive with no other viral (A, B, and E) co-infections as determined by ELISA. Also, the RT-PCR detected HCV RNA in these ELISA positive children. The remaining children (179/184) were all negative for all hepatitis viruses' markers (HAV IgM, HEV IgM, HBs Ag, and HCV Ab). In conclusion, this study documented that, Cairo hospitals serving Egyptian children had a low prevalence of viral hepatitis (A, B, C, and E). More research with larger sample sizes from hospitals across Egypt is needed.

Effect of temperature on hepatitis a virus and exploration of binding mode mechanism of phytochemicals from tinospora cordifolia: an insight into molecular docking, MM/GBSA, and molecular dynamics simulation study.

The hepatitis A virus (HAV), which causes hepatitis A, is a contagious liver ailment. The infections are not specifically treated by any medications. Therefore, the development of less harmful, more effective and cost-effective antiviral agents are necessary. The present work highlighted the in-silico activity of phytocompounds from tinospora cordifolia against HAV. The binding interaction of HAV with the phytocompounds was analyzed through molecular docking. Molecular docking revealed that chasmanthin, malabarolide, menispermacide, tinosporaside, and tinosporinone compounds bind with HAV more efficiently than other compounds. Further evaluation using 100 ns molecular dynamics simulation, MM/GBSA and free energy landscape indicated that all phytocompounds studied here were found to be most promising drug candidate against hepatitis A virus. Our computational study will encourage promoting in further investigation for in vitro and in vivo clinical trials.Communicated by Ramaswamy H. Sarma.

Determining Herd Immunity Thresholds for Hepatitis A Virus Transmission to Inform Vaccination Strategies Among People Who Inject Drugs in 16†US States.

BACKGROUND: Widespread outbreaks of person-to-person transmitted hepatitis A virus (HAV), particularly among people who inject drugs (PWID), continue across the United States and globally. However, the herd immunity threshold and vaccination coverage required to prevent outbreaks are unknown. We used surveillance data and dynamic modeling to estimate herd immunity thresholds among PWID in 16 US states. METHODS: We used a previously published dynamic model of HAV transmission calibrated to surveillance data from outbreaks involving PWID in 16 states. Using state-level calibrated models, we estimated the basic reproduction number (R0) and herd immunity threshold for PWID in each state. We performed a meta-analysis of herd immunity thresholds to determine the critical vaccination coverage required to prevent most HAV outbreaks among PWID. RESULTS: Estimates of R0 for HAV infection ranged from 2.2 (95% confidence interval [CI], 1.9-2.5) for North Carolina to 5.0 (95% CI, 4.5-5.6) for West Virginia. Corresponding herd immunity thresholds ranged from 55% (95% CI, 47%-61%) for North Carolina to 80% (95% CI, 78%-82%) for West Virginia. Based on the meta-analysis, we estimated a pooled herd immunity threshold of 64% (95% CI, 61%-68%; 90% prediction interval, 52%-76%) among PWID. Using the prediction interval upper bound (76%) and assuming 95% vaccine efficacy, we estimated that vaccination coverage of 80% could prevent most HAV outbreaks. CONCLUSIONS: Hepatitis A vaccination programs in the United States may need to achieve vaccination coverage of at least 80% among PWID in order to prevent most HAV outbreaks among this population.

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.