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.

Drug Screening for Hepatitis A Virus (HAV): Nicotinamide Inhibits c-Jun Expression and HAV Replication.

Hepatitis A virus (HAV) infection often causes acute hepatitis, which results in a case fatality rate of 0.2% and fulminant hepatitis in 0.5% of cases. However, no specific potent anti-HAV drug is available on the market to date. In the present study, we focused on inhibition of HAV internal ribosomal entry site (IRES)-mediated translation and investigated novel therapeutic drugs through drug repurposing by screening for inhibitors of HAV IRES-mediated translation and cell viability using a reporter assay and cell viability assay, respectively. The initial screening of 1,158 drugs resulted in 77 candidate drugs. Among them, nicotinamide significantly inhibited HAV HA11-1299 genotype IIIA replication in Huh7 cells. This promising drug also inhibited HAV HM175 genotype IB subgenomic replicon and HAV HA11-1299 genotype IIIA replication in a dose-dependent manner. In the present study, we found that nicotinamide inhibited the activation of activator protein 1 (AP-1) and that knockdown of c-Jun, which is one of the components of AP-1, inhibited HAV HM175 genotype IB IRES-mediated translation and HAV HA11-1299 genotype IIIA and HAV HM175 genotype IB replication. Taken together, the results showed that nicotinamide inhibited c-Jun, resulting in the suppression of HAV IRES-mediated translation and HAV replication, and therefore, it could be useful for the treatment of HAV infection. IMPORTANCE Drug screening methods targeting HAV IRES-mediated translation with reporter assays are attractive and useful for drug repurposing. Nicotinamide (vitamin B3, niacin) has been shown to effectively inhibit HAV replication. Transcription complex activator protein 1 (AP-1) plays an important role in the transcriptional regulation of cellular immunity or viral replication. The results of this study provide evidence that AP-1 is involved in HAV replication and plays a role in the HAV life cycle. In addition, nicotinamide was shown to suppress HAV replication partly by inhibiting AP-1 activity and HAV IRES-mediated translation. Nicotinamide may be useful for the control of acute HAV infection by inhibiting cellular AP-1 activity during HAV infection processes.

Macrophage Depletion Reactivates Fecal Virus Shedding following Resolution of Acute Hepatitis A in Ifnar1-/- Mice.

Although hepatitis A virus (HAV) is associated only with acute hepatitis in humans, HAV RNA persists within the liver for months following resolution of liver inflammation and cessation of fecal virus shedding in chimpanzees and murine models of hepatitis A. Here, we confirm striking differences in the kinetics of HAV RNA clearance from liver versus serum and feces in infected Ifnar1-/- mice and investigate the nature of viral RNA persisting in the liver following normalization of serum alanine aminotransferase (ALT) levels. Fecal shedding of virus produced in hepatocytes declined >3,000-fold between its peak at day 14 and day 126, whereas intrahepatic HAV RNA declined only 32-fold by day 154. Viral RNA was identified within hepatocytes 3 to 4 months after inoculation and was associated with membranes, banding between 1.07 and 1.14 g/cm3 in isopycnic iodixanol gradients. Gradient fractions containing HAV RNA demonstrated no infectivity when inoculated into naive mice but contained neutralizing anti-HAV antibody. Depleting CD4+ or CD8+ T cells at this late point in infection had no effect on viral RNA abundance in the liver, whereas clodronate-liposome depletion of macrophages between days 110 and 120 postinoculation resulted in a striking recrudescence of fecal virus shedding and the reappearance of viral RNA in serum coupled with reductions in intra-hepatic Ifnγ, Tnfα, Ccl5, and other chemokine transcripts. Our data suggest that replication-competent HAV RNA persists for months within the liver in the presence of neutralizing antibody following resolution of acute hepatitis in Ifnar1-/- mice and that macrophages play a key role in viral control late in infection. IMPORTANCE HAV RNA persists in the liver of infected chimpanzees and interferon receptor-deficient Ifnar1-/- mice for many months after neutralizing antibodies appear, virus has been cleared from the blood, and fecal virus shedding has terminated. Here, we show this viral RNA is located within hepatocytes and that the depletion of macrophages months after the resolution of hepatic inflammation restores fecal virus shedding and circulating viral RNA. Our study identifies an important role for macrophages in virus control following resolution of acute hepatitis A in Ifnar1-/- mice and may have relevance to relapsing hepatitis A in humans.

Nonlytic Quasi-Enveloped Hepatovirus Release Is Facilitated by pX Protein Interaction with the E3 Ubiquitin Ligase ITCH.

Hepatoviruses are atypical hepatotropic picornaviruses that are released from infected cells without lysis in small membranous vesicles. These exosome-like, quasi-enveloped virions (eHAV) are infectious and the only form of hepatitis A virus (HAV) found circulating in blood during acute infection. eHAV is released through multivesicular endosomes in a process dependent on endosomal sorting complexes required for transport (ESCRT). Capsid protein interactions with the ESCRT-associated Bro1 domain proteins, ALG-2-interacting protein X (ALIX) and His domain-containing protein tyrosine phosphatase (HD-PTP), which are both recruited to the pX domain of 1D (VP1pX), are critical for this process. Previous proteomics studies suggest pX also binds the HECT domain, NEDD4 family E3 ubiquitin ligase, ITCH. Here, we confirm this interaction and show ITCH binds directly to the carboxy-terminal half of pX from both human and bat hepatoviruses independently of ALIX. A small chemical compound (compound 5) designed to disrupt interactions between WW domains of NEDD4 ligases and substrate molecules blocked ITCH binding to pX and demonstrated substantial antiviral activity against HAV. CRISPR deletion or small interfering RNA (siRNA) knockdown of ITCH expression inhibited the release of a self-assembling nanocage protein fused to pX and also impaired the release of eHAV from infected cells. The release could be rescued by overexpression of wild-type ITCH, but not a catalytically inactive ITCH mutant. Despite this, we found no evidence that ITCH ubiquitylates pX or that eHAV release is strongly dependent upon Lys residues in pX. These data indicate ITCH plays an important role in the ESCRT-dependent release of quasi-enveloped hepatovirus, although the substrate molecule targeted for ubiquitylation remains to be determined. IMPORTANCE Mechanisms underlying the cellular release of quasi-enveloped hepatoviruses are only partially understood, yet play a crucial role in the pathogenesis of this common agent of viral hepatitis. Multiple NEDD4 family E3 ubiquitin ligases, including ITCH, have been reported to promote the budding of conventional enveloped viruses but are not known to function in the release of HAV or other picornaviruses from infected cells. Here, we show that the unique C-terminal pX extension of the VP1 capsid protein of HAV interacts directly with ITCH and that ITCH promotes eHAV release in a manner analogous to its role in budding of some conventional enveloped viruses. The catalytic activity of ITCH is required for efficient eHAV release and may potentially function to ubiquitylate the viral capsid or activate ESCRT components.

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.

A genome-wide CRISPR screen identifies UFMylation and TRAMP-like complexes as host factors required for hepatitis A virus infection.

Hepatitis A virus (HAV) is a positive-sense RNA virus causing acute inflammation of the liver. Here, using a genome-scale CRISPR screen, we provide a comprehensive picture of the cellular factors that are exploited by HAV. We identify genes involved in sialic acid/ganglioside biosynthesis and members of the eukaryotic translation initiation factor complex, corroborating their putative roles for HAV. Additionally, we uncover all components of the cellular machinery for UFMylation, a ubiquitin-like protein modification. We show that HAV translation specifically depends on UFM1 conjugation of the ribosomal protein RPL26. Furthermore, we find that components related to the yeast Trf4/5-Air1/2-Mtr4 polyadenylation (TRAMP) complex are required for viral translation independent of controlling viral poly(A) tails or RNA stability. Finally, we demonstrate that pharmacological inhibition of the TRAMP-like complex decreases HAV replication in hepatocyte cells and human liver organoids, thus providing a strategy for host-directed therapy of HAV infection.

Electron Beam Susceptibility of Enteric Viruses and Surrogate Organisms on Fruit, Seed and Spice Matrices.

The objective of this study was to use high-energy electron beam (HEEB) treatments to find surrogate microorganisms for enteric viruses and to use the selected surrogates as proof of concept to investigate low-energy electron beam (LEEB) treatments for enteric virus inactivation at industrial scale on frozen blueberries. Six food matrices inoculated with HAV (hepatitis A virus), MNV S99 (murine norovirus), bacteriophages MS2 and Qß, and Geobacillus stearothermophilus spores were treated with HEEB at 10 MeV using 4, 8 and 16 kGy doses. G. stearothermophilus spores showed the highest inactivation on all matrices except on raisins, with a dose-dependent effect. HAV reached the maximum measurable log10 reduction (> 3.2 log10) when treated at 16 kGy on raisins. MNV showed the highest resistance of all tested microorganisms, independent of the dose, except on frozen blueberries. On frozen blueberries, freeze-dried raspberries, sesame seeds and black peppercorns, HAV showed a mean inactivation level in between those of MS2 and G. stearothermophilus. Based on this, we selected both surrogate organisms as first approximation to estimate HAV inactivation on frozen blueberries during LEEB treatment at 250 keV using 16 kGy. Reductions of 3.1 and 1.3 log10 were measured for G. stearothermophilus spores and MS2, respectively, suggesting that a minimum reduction of 1.4 log10 can be expected for HAV under the same conditions.

Survival and Inactivation by Advanced Oxidative Process of Foodborne Viruses in Model Low-Moisture Foods.

Enteric viruses, such as human norovirus (NoV) and hepatitis A virus (HAV), are the major causes of foodborne illnesses worldwide. These viruses have low infectious dose, and may remain infectious for weeks in the environment and food. Limited information is available regarding viral survival and transmission in low-moisture foods (LMF). LMFs are generally considered as ready-to-eat products, which undergo no or minimal pathogen reduction steps. However, numerous foodborne viral outbreaks associated with LMFs have been reported in recent years. The objective of this study was to examine the survival of foodborne viruses in LMFs during 4-week storage at ambient temperature and to evaluate the efficacy of advanced oxidative process (AOP) treatment in the inactivation of these viruses. For this purpose, select LMFs such as pistachios, chocolate, and cereal were inoculated with HAV and the norovirus surrogates, murine norovirus (MNV) and feline calicivirus (FCV), then viral survival on these food matrices was measured over a four-week incubation at ambient temperature, by both plaque assay and droplet-digital RT-PCR (ddRT-PCR) using the modified ISO-15216 method as well as the magnetic bead assay for viral recovery. We observed an approximately 0.5 log reduction in viral genome copies, and 1 log reduction in viral infectivity for all three tested viruses following storage of select inoculated LMFs for 4 weeks. Therefore, the present study shows that the examined foodborne viruses can persist for a long time in LMFs. Next, we examined the inactivation efficacy of AOP treatment, which combines UV-C, ozone, and hydrogen peroxide vapor, and observed that while approximately 100% (4 log) inactivation can be achieved for FCV, and MNV in chocolate, the inactivation efficiency diminishes to approximately 90% (1 log) in pistachios and 70% (< 1 log) in cereal. AOP treatment could therefore be a good candidate for risk reduction of foodborne viruses from certain LMFs depending on the food matrix and surface of treatment.

Detection of norovirus, hepatitis A and hepatitis E viruses in multicomponent foodstuffs.

Among the enteric viruses implicated in foodborne outbreaks, the human norovirus and hepatitis viruses A and E (HAV and HEV) represent a serious public health concern. International standard ISO 15216 proposes methods for detecting HAV and norovirus (genogroups I and II) RNA from soft fruit, leaf, stem and bulb vegetables, bottled water or food surfaces. These methods had not previously been validated for detecting the targeted viruses in other foodstuffs such as multicomponent foods, nor for detecting other viruses in foodstuffs. The aim of this study was to characterise a method derived from the vegetable method described in ISO 15216 to detect HAV, HEV and norovirus in artificially-contaminated multicomponent foodstuffs according to the recent international standard ISO 16140-4. Results showed that the mean recovery rates for all settings did not differ according to the operator. The mean extraction yields ranged from 0.35% to 40.44% for HAV, 5.19% to 100% for HEV, 0.10% to 40.61% for norovirus GI and 0.88% to 69.16% for norovirus GII. The LOD95 was 102 genome copies/g for HAV, HEV and norovirus GII and 103 genome copies/g for norovirus GI. The LOQ was 2.90 × 104, 1.40 × 103, 1.60 × 104 and 1.30 × 104 genome copies/g for HAV, HEV, norovirus GI and norovirus GII respectively. The MNV-1 process control was detected in 120 out of 128 RNA extracts analysed and was recovered with an efficiency of between 3.83% and 50.22%. The mean inhibition rates of quantitative real-time RT-PCR reaction ranged from 3.25% to 28.70% and varied significantly with the type of food matrix. The described method could be used to detect viruses in composite food products for routine diagnosis needs.

Inactivation of hepatitis A virus and murine norovirus on surfaces of plastic, steel and raspberries using steam-ultrasound treatment.

The leading causes of foodborne viral disease outbreaks are human norovirus and hepatitis A virus (HAV). Their environmental persistence enables contamination of kitchen surfaces and crops often consumed raw, such as berries. Many decontamination procedures are inefficient and unsuitable for surfaces of industrial kitchen environments and soft fruits. In this study, we investigated the efficiency of a novel surface decontamination technology, combining steam and ultrasound (steam-ultrasound). Plastic, steel or raspberry surfaces were spiked with the norovirus surrogate, murine norovirus (MNV), and HAV, and steam-ultrasound treated at 85, 90 and 95 °C for 0-5 s. Post treatment viruses were titrated for survival by plaque assay and for genome stability by real-time quantitative PCR (RT-qPCR) of nucleic acid extracts. Survival of viruses were estimated in a log-linear model and the treatment time requirements for each decimal reduction (D value) in viral survival were calculated. The estimated D values of MNV or HAV were 0.4-0.2 or 1.1-0.8 s on plastic, 0.9-0.7 or 1.4-0.8 s on steel and 1.6-1.7 or 3.2-4.7 s on raspberries. No clear trend of genome reduction was observed with tested treatment parameters. Raspberries treated up to 4 s retained its natural texture and visual appeal similar to untreated controls whilst monitored for 7 days. In conclusion, steam-ultrasound treatment can within seconds reduce the titre of foodborne viruses on surfaces of plastic, steel and raspberries. This may particularly benefit industrial scale production of soft fruits for raw consumption and for swift non-hazardous decontamination of industrial kitchen surfaces.

Thermal Inactivation of Hepatitis A Virus, Noroviruses, and Simian Rotavirus in Cows' Milk.

Consumption of raw or unpasteurized milk is a risk for the consumers because indirect contaminations such as fecal-cross-contamination could occur and determine the presence of enteric viruses. In this study, milk was treated with several temperature and time combinations chosen by performing a preliminary experiment to evaluate the intervals needed to inactivate Hepatitis A virus (HAV) HM175 strain, noroviruses genogroups I and II (GI and GII), and simian rotavirus SA11 at different temperatures. Results were obtained by measuring the genome copies and infectious units by real-time PCR and plaque assays respectively. At 85 °C, one minute and two minutes were needed to achieve 6.6 log10 ± 0.2 and 8 log10 ± 0 reductions of genome copies of HAV respectively. Similar genome copies reduction was also observed for noroviruses (GI and GII) and simian rotavirus. At higher temperatures, 90 s (s) at 90 °C and 60 s at 95 °C were needed to achieve 8 log10 ± 0 reductions of the genome copies of all studied viruses. Significant higher sensitivity of the infectious units of both HAV and simian rotavirus to heat treatment of milk than their genome copies was observed. At boiling point of milk (100.5 °C), 40 s were needed to achieve 8 log10 ± 0 reductions of genome copies of all the studied viruses, while 10 s were needed to achieve 6 log10 ± 0 reductions of the infectious units of HAV and simian rotavirus. Significant higher reduction of infectious units than genome copies was observed confirming that genome copies reduction does not correspond to infectious virus.

Green tea extract assisted low-temperature pasteurization to inactivate enteric viruses in juices.

The current popularity of minimally processed foods is an opportunity for natural antimicrobial agents to be combined with mild heat treatments to act synergistically in reducing viral foodborne pathogens. Viral inactivation by heat-treatments (at 25, 40, 50 and 63 °C for 30 min) combined with aged green tea extract (aged-GTE) was initially evaluated in phosphate buffered saline (PBS) against murine norovirus (MNV-1) and hepatitis A virus (HAV) by cell culture, and against human norovirus by in situ capture RT-qPCR. The combination of aged-GTE and heat treatment at 50 °C for 30 min exerted strong antiviral activity, reducing by more than 5 log MNV-1 infectivity in PBS. Heating at 40 °C for 30 min reduced the binding of norovirus to porcine gastric mucine (PGM) to 41.5% and the addition of aged-GTE further decreased the binding to 4.7%. Additionally, the reduction of MNV-1 and HAV infectivity was investigated in two different types of juices exposed to mild heat treatments alone, and combined with aged-GTE. The addition of aged-GTE increased to more than 4 log the inactivation of MNV-1 in juices exposed to 50 °C for 30 min. However, this synergistic effect of aged-GTE combined with heat treatments was not observed for HAV in any of the juices. Aged-GTE, then, could be considered as an additional control measure to improve the food safety of mild heat pasteurized juices.

Cell Culture Systems and Drug Targets for Hepatitis A Virus Infection.

Hepatitis A virus (HAV) infection is one of the major causes of acute hepatitis, and this infection occasionally causes acute liver failure. HAV infection is associated with HAV-contaminated food and water as well as sexual transmission among men who have sex with men. Although an HAV vaccine has been developed, outbreaks of hepatitis A and life-threatening severe HAV infections are still observed worldwide. Therefore, an improved HAV vaccine and anti-HAV drugs for severe hepatitis A should be developed. Here, we reviewed cell culture systems for HAV infection, and other issues. This review may help with improving the HAV vaccine and developing anti-HAV drugs.

Hepatitis A infections from food.

Food contaminated by hepatitis A virus (HAV) is responsible of the 2-7% of all HAV outbreaks worldwide. This review provides a description of the HAV characteristics, its infectivity and epidemiological features. In addition, this review compiles existing original papers reporting HAV prevalence, viral titres in foodstuffs and the risk associated with food contamination. The purpose of this revision is to conduct a structured and systematic review of the published molecular procedures for HAV detection in food, including the assessment of its infectivity.

Heat sensitization of hepatitis A virus and Tulane virus using grape seed extract, gingerol and curcumin.

Human noroviruses (HNoV) and hepatitis A virus (HAV) are predominantly linked to foodborne outbreaks worldwide. As cell-culture systems to propagate HNoV in laboratories are not easily available, Tulane virus (TV) is used as a cultivable HNoV surrogate to determine inactivation. Heat-sensitization of HAV and TV by "generally recognized as safe'' (GRAS) substances can potentially reduce their time-temperature inactivation parameters during processing to ensure food safety. Curcumin, gingerol (from ginger), and grape seed extract (GSE) reportedly have anti-inflammatory, immune-modulating and antiviral properties. The objective of this study was to determine and compare the D-values and z-values of HAV and TV at 52-68 °C with or without curcumin (0.015 mg/ml), gingerol (0.1 mg/ml), or GSE (1 mg/ml) in 2-ml glass vials. HAV at ~7 log PFU/ml and TV at ~6 log PFU/ml were diluted in phosphate buffered saline (PBS) and added to two sets of six 2-mL sterile glass vials. One set served as the control and the second set had the three extracts individually added for thermal treatments in a circulating water bath for 0-10 min. The D-values for TV in PBS ranged from 4.55 ± 0.28 to 1.08 ± 0.16 min, and for HAV in PBS ranged from to 9.21 ± 0.24 to 0.67 ± 0.19 min at 52-68 °C. Decreased D-values (52-58 °C) for TV with curcumin ranging from 4.32 ± 0.25 to 0.62 ± 0.17 min, gingerol from 4.09 ± 0.18 to 0.72 ± 0.09 min and GSE from 3.82 ± 0.18 to 0.80 ± 0.07 min, with similar trends for HAV were observed. The linear model showed significant differences (p < 0.05) between the D-values of HAV and TV with and without plant extracts for most tested temperatures. This suggests that GRAS substances can potentially lower temperature and time regimens needed to inactivate HAV and TV.

Fetal Calf Serum Exerts an Inhibitory Effect on Replication of Duck Hepatitis A Virus Genotype 1 in Duck Embryo Fibroblast Cells.

Among the causative agents of duck viral hepatitis, duck hepatitis A virus genotype 1 (DHAV-1) is the most common virus reported in most outbreaks worldwide. How to propagate DHAV-1 in cell cultures efficiently remains a problem to be explored. Here, we aimed to test the effect of serum type on DHAV-1 replication in duck embryo fibroblast (DEF) cells. Comparative studies involved virus culture and passage, observation of cytopathic effect (CPE), virus quantification, and plaque formation assay. From the results of these investigations, we conclude that use of chicken serum (CS) in maintenance medium allows DHAV-1 to establish productive, cytocidal infection in DEF cells, whereas FCS exerts inhibitory effects on DHAV-1 replication, CPE development, and plaque formation. By using a neutralization test, we found that the direct action of FCS on virions is likely to play a key role in inhibiting DHAV-1 replication in DEF cells. Mechanism analyses revealed that FCS inhibits DHAV-1 replication at virus adsorption and reduces extracellular virus yields. The present work may shed light on a new perspective for antiviral agent development, and have provided a virus-host cell system for further studies on molecular mechanism involved DHAV-1 replication and pathogenesis.

Quasi-enveloped hepatitis virus assembly and release.

Hepatitis A virus (HAV) and hepatitis E virus (HEV) infections are the main causes for acute hepatitis worldwide. Both viruses had long been considered as nonenveloped viruses. However, recent work has uncovered that both viruses circulate in the bloodstream as membrane-cloaked, "quasi-enveloped" particles that are, surprisingly, infectious and likely the only form mediating virus spread within the host. The discovery of quasi-enveloped HAV and HEV particles has fundamentally changed the traditional view on the life cycle and pathogenesis of these viruses. However, because HAV and HEV are phylogenetically unrelated and their capsid assembly processes are quite distinct, it is not clear whether they use similar or different mechanisms for envelopment and exit. This review provides an overview of the current knowledge about the assembly and exit processes of HAV and HEV and perspectives for future studies.

Advances in antiviral polysaccharides derived from edible and medicinal plants and mushrooms.

Despite the number of approved antivirals has considerably increased, these existing drugs are not always efficacious or well-tolerated and drug-resistant virus strains are rapidly emerging. Nowadays, many polysaccharides as independent or the main bioactive ingredients have been approved as medicines. The present report aims to provide systematically reorganized information on antiviral polysaccharides derived from edible and medicinal plants and mushrooms (PsEMPM) to people for better utilization of them. PsEMPM can inhibit the infection of viruses by interfering with a few steps in the virus life cycle and/or improving the host antiviral immune responses. Polyanionic nature and sulfates are in many cases required antiviral potency of PsEMPM, while it not only is a function of high charge density but also associated with distinct structural specificities. Plenty of efforts have been devoted to achieving the discovery of novel antiviral polysaccharides, however, the detailed structural characteristics of PsEMPM still merit further in-depth investigation.

Emerging hepatitis E virus compared with hepatitis A virus: A new sanitary challenge.

Hepatitis A (HAV) and E (HEV) viruses are able to cause liver disease in humans. Among the five classical hepatotropic viruses, they are mainly transmitted via the fecal-oral route. Historically, many similarities have thus been described between them according to their incidence and their pathogenicity, especially in countries with poor sanitary conditions. However, recent advances have provided new insights, and the gap is widening between them. Indeed, while HAV infection incidence tends to decrease in developed countries along with public health improvement, HEV is currently considered as an underdiagnosed emerging pathogen. HEV autochthonous infections are increasingly observed and are mainly associated with zoonotic transmissions. Extra hepatic signs resulting in neurological or renal impairments have also been reported for HEV, as well as a chronic carrier state in immunocompromised patients, arguing in favor of differential pathogenesis between those two viruses. Recent molecular tools have allowed studies of viral genome variability and investigation of links between viral plasticity and clinical evolution. The identification of key functional mutations in viral genomes may improve the knowledge of their clinical impact and is analyzed in depth in the present review.

The Critical Role of Codon Composition on the Translation Efficiency Robustness of the Hepatitis A Virus Capsid.

Hepatoviruses show an intriguing deviated codon usage, suggesting an evolutionary signature. Abundant and rare codons in the cellular genome are scarce in the human hepatitis A virus (HAV) genome, while intermediately abundant host codons are abundant in the virus. Genotype-phenotype maps, or fitness landscapes, are a means of representing a genotype position in sequence space and uncovering how genotype relates to phenotype and fitness. Using genotype-phenotype maps of the translation efficiency, we have shown the critical role of the HAV capsid codon composition in regulating translation and determining its robustness. Adaptation to an environmental perturbation such as the artificial induction of cellular shutoff-not naturally occurring in HAV infection-involved movements in the sequence space and dramatic changes of the translation efficiency. Capsid rare codons, including abundant and rare codons of the cellular genome, slowed down the translation efficiency in conditions of no cellular shutoff. In contrast, rare capsid codons that are abundant in the cellular genome were efficiently translated in conditions of shutoff. Capsid regions very rich in slowly translated codons adapt to shutoff through sequence space movements from positions with highly robust translation to others with diminished translation robustness. These movements paralleled decreases of the capsid physical and biological robustness, and resulted in the diversification of capsid phenotypes. The deviated codon usage of extant hepatoviruses compared with that of their hosts may suggest the occurrence of a virus ancestor with an optimized codon usage with respect to an unknown ancient host.