Role of Disulphide Bonds in Membrane Partitioning of a Viral Peptide.

The importance of disulphide bond in mediating viral peptide entry into host cells is well known. In the present work, we elucidate the role of disulphide (SS) bond in partitioning mechanism of membrane-active Hepatitis A Virus-2B (HAV-2B) peptide, which harbours three cysteine residues promoting formation of multiple SS-bonded states. The inclusion of SS-bond not only results in a compact conformation but also induces distorted α-helical hairpin geometry in comparison to SS-free state. Owing to these, the hydrophobic residues get buried, restricting the insertion of SS-bonded HAV-2B peptide into lipid packing defects and thus the partitioning of the peptide is completely or partly abolished. In this way, the disulphide bond can potentially regulate the partitioning of HAV-2B peptide such that the membrane remodelling effects of this viral peptide are significantly reduced. The current findings may have potential implications in drug designing, targeting the HAV-2B protein by promoting disulphide bond formation within its membrane-active region.

Glass Wool Concentration Optimization for the Detection of Enveloped and Non-enveloped Waterborne Viruses.

An extremely affordable virus concentration method based on adsorption-elution to glass wool and subsequent reconcentration through polyethylene glycol 6000 (PEG) precipitation was optimized to recover not only non-enveloped viruses but also enveloped viruses. Hepatitis A virus (HAV) and transmissible gastroenteritis virus (TGEV) were employed as surrogates for naked and enveloped viruses, respectively, to set up the methodology. Initial experimentation in small-volume samples showed that both types of particles readily adsorbed to the positively charged glass wool but were poorly detached from it through standard elution with 0.05 M glycine with 3% of beef extract buffer, pH 9.5, with elution efficiencies of 7.2% and 2.6%, for HAV and TGEV, respectively. To improve the recovery of enveloped viruses, several modifications in the elution were assayed: increasing the elution pH, extending glass wool and eluent contact time, adding a detergent, or performing the elution by recirculation or under agitation. Considering practicability and performance, recircularization of the eluent at pH 11.0 for 20 min was the elution procedure of choice, with efficiencies of 25.7% and 18.8% for HAV and TGEV in 50 L of water. Additionally, employing 20% PEG instead of 10% for virus reconcentration improved recoveries up to 47% and 51%, respectively. The optimized procedure was applied to detect naturally occurring HAV and coronaviruses in surface water of Wadi Hanifa, Riyadh. HAV was detected in 38% of the samples, while one sample was positive for an alphacoronavirus. This cheap virus detection system enables the comprehensive surveillance of viruses present in water samples.

Hepatitis A Virus Capsid Structure.

Hepatitis A virus (HAV) has been enigmatic, evading detailed structural analysis for many years. Its recently determined high-resolution structure revealed an angular surface without the indentations often characteristic of receptor-binding sites. The viral protein 1 (VP1) ß-barrel shows no sign of a pocket factor and the amino terminus of VP2 displays a "domain swap" across the pentamer interface, as in a subset of mammalian picornaviruses and insect picorna-like viruses. Structure-based phylogeny confirms this placement. These differences suggest an uncoating mechanism distinct from that of enteroviruses. An empty capsid structure reveals internal differences in VP0 and the VP1 amino terminus connected with particle maturation. An HAV/Fab complex structure, in which the antigen-binding fragment (Fab) appears to act as a receptor-mimic, clarifies some historical epitope mapping data, but some remain difficult to reconcile. We still have little idea of the structural features of enveloped HAV particles.

Enzymatic and viability RT-qPCR assays for evaluation of enterovirus, hepatitis A virus and norovirus inactivation: Implications for public health risk assessment.

AIM: To assess the potential of a viability dye and an enzymatic reverse transcription quantitative PCR (RT-qPCR) pretreatment to discriminate between infectious and noninfectious enteric viruses. METHODS AND RESULTS: Enterovirus (EntV), norovirus (NoV) GII.4 and hepatitis A virus (HAV) were inactivated at 95°C for 10 min, and four methods were used to compare the efficiency of inactivation: (i) cell culture plaque assay for HAV and EntV, (ii) RT-qPCR alone, (iii) RT-qPCR assay preceded by RNase treatment, and (iv) pretreatment with a viability dye (reagent D (RD)) followed by RT-qPCR. In addition, heat-inactivated NoV was treated with RD coupled with surfactants to increase the efficiency of the viability dye. No treatment was able to completely discriminate infectious from noninfectious viruses. RD-RT-qPCR reduced more efficiently the detection of noninfectious viruses with little to no removal observed with RNase. RD-RT-qPCR method was the closest to cell culture assay. The combination of surfactants and RD did not show relevant improvements on the removal of inactivated viruses signal compared with viability RT-qPCR, with the exception of Triton X-100. CONCLUSION: The use of surfactant/RD-RT-qPCR, although not being able to completely remove the signal from noninfectious viral particles, yielded a better estimation of viral infectivity. SIGNIFICANCE AND IMPACT OF THE STUDY: Surfactant/RD-RT-qPCR may be an advantageous tool for a better detection of infectious viruses with potential significant impact in the risk assessment of the presence of enteric viruses.

Detection of Potential Infectious Enteric Viruses in Fresh Produce by (RT)-qPCR Preceded by Nuclease Treatment.

Foodborne illnesses associated with contaminated fresh produce are a common public health problem and there is an upward trend of outbreaks caused by enteric viruses, especially human noroviruses (HNoVs) and hepatitis A virus (HAV). This study aimed to assess the use of DNase and RNase coupled to qPCR and RT-qPCR, respectively, to detect intact particles of human adenoviruses (HAdVs), HNoV GI and GII and HAV in fresh produce. Different concentrations of DNase and RNase were tested to optimize the degradation of free DNA and RNA from inactivated HAdV and murine norovirus (MNV), respectively. Results indicated that 10 µg/ml of RNase was able to degrade more than 4 log10 (99.99%) of free RNA, and 1 U of DNase degraded the range of 0.84-2.5 log10 of free DNA depending on the fresh produce analysed. The treatment with nucleases coupled to (RT)-qPCR was applied to detect potential infectious virus in organic lettuce, green onions and strawberries collected in different seasons. As a result, no intact particles of HNoV GI and GII were detected in the 36 samples analysed, HAdV was found in one sample and HAV was present in 33.3% of the samples, without any reasonable distribution pattern among seasons. In conclusion, RT-qPCR preceded by RNase treatment of eluted samples from fresh produce is a good alternative to detect undamaged RNA viruses and therefore, potential infectious viruses. Moreover, this study provides data about the prevalence of enteric viruses in organic fresh produce from Brazil.

Solubility as a limiting factor for expression of hepatitis A virus proteins in insect cell-baculovirus system.

The use of recombinant proteins may represent an alternative model to inactivated vaccines against hepatitis A virus (HAV). The present study aimed to express the VP1 protein of HAV in baculovirus expression vector system (BEVS). The VP1 was expressed intracellularly with molecular mass of 35 kDa. The VP1 was detected both in the soluble fraction and in the insoluble fraction of the lysate. The extracellular expression of VP1 was also attempted, but the protein remained inside the cell. To verify if hydrophobic characteristics would also be present in the HAV structural polyprotein, the expression of P1-2A protein was evaluated. The P1-2A polyprotein remained insoluble in the cellular extract, even in the early infection stages. These results suggest that HAV structural proteins are prone to form insoluble aggregates. The low solubility represents a drawback for production of large amounts of HAV proteins in BEVS.

Solubility as a limiting factor for expression of hepatitis A virus proteins in insect cell-baculovirus system

The use of recombinant proteins may represent an alternative model to inactivated vaccines against hepatitis A virus (HAV). The present study aimed to express the VP1 protein of HAV in baculovirus expression vector system (BEVS). The VP1 was expressed intracellularly with molecular mass of 35 kDa. The VP1 was detected both in the soluble fraction and in the insoluble fraction of the lysate. The extracellular expression of VP1 was also attempted, but the protein remained inside the cell. To verify if hydrophobic characteristics would also be present in the HAV structural polyprotein, the expression of P1-2A protein was evaluated. The P1-2A polyprotein remained insoluble in the cellular extract, even in the early infection stages. These results suggest that HAV structural proteins are prone to form insoluble aggregates. The low solubility represents a drawback for production of large amounts of HAV proteins in BEVS.

Thermal inactivation kinetics of hepatitis A virus in homogenized clam meat (Mercenaria mercenaria).

AIMS: Epidemiological evidence suggests that hepatitis A virus (HAV) is the most common pathogen transmitted by bivalve molluscs such as clams, cockles, mussels and oysters. This study aimed to generate thermal inactivation kinetics for HAV as a first step to design adequate thermal processes to control clam-associated HAV outbreaks. METHODS AND RESULTS: Survivor curves and thermal death curves were generated for different treatment times (0-6 min) at different temperatures (50-72°C) and Weibull and first-order models were compared. D-values for HAV ranged from 47·37 ± 1·23 to 1·55 ± 0·12 min for the first-order model and 64·43 ± 3·47 to 1·25 ± 0·45 min for the Weibull model at temperatures from 50 to 72°C. z-Values for HAV in clams were 12·97 ± 0·59°C and 14·83 ± 0·0·28°C using the Weibull and first-order model respectively. The calculated activation energies for the first-order and Weibull model were 145 and 170 kJ mole(-1) respectively. CONCLUSION: The Weibull model described the thermal inactivation behaviour of HAV better than the first-order model. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides novel and precise information on thermal inactivation kinetics of HAV in homogenized clams. This will enable reliable thermal process calculations for HAV inactivation in clams and closely related seafood.

Thermal Inactivation Kinetics of Human Norovirus Surrogates and Hepatitis A Virus in Turkey Deli Meat.

Human noroviruses (HNoV) and hepatitis A virus (HAV) have been implicated in outbreaks linked to the consumption of presliced ready-to-eat deli meats. The objectives of this research were to determine the thermal inactivation kinetics of HNoV surrogates (murine norovirus 1 [MNV-1] and feline calicivirus strain F9 [FCV-F9]) and HAV in turkey deli meat, compare first-order and Weibull models to describe the data, and calculate Arrhenius activation energy values for each model. The D (decimal reduction time) values in the temperature range of 50 to 72°C calculated from the first-order model were 0.1 ± 0.0 to 9.9 ± 3.9 min for FCV-F9, 0.2 ± 0.0 to 21.0 ± 0.8 min for MNV-1, and 1.0 ± 0.1 to 42.0 ± 5.6 min for HAV. Using the Weibull model, the tD = 1 (time to destroy 1 log) values for FCV-F9, MNV-1, and HAV at the same temperatures ranged from 0.1 ± 0.0 to 11.9 ± 5.1 min, from 0.3 ± 0.1 to 17.8 ± 1.8 min, and from 0.6 ± 0.3 to 25.9 ± 3.7 min, respectively. The z (thermal resistance) values for FCV-F9, MNV-1, and HAV were 11.3 ± 2.1°C, 11.0 ± 1.6°C, and 13.4 ± 2.6°C, respectively, using the Weibull model. The z values using the first-order model were 11.9 ± 1.0°C, 10.9 ± 1.3°C, and 12.8 ± 1.7°C for FCV-F9, MNV-1, and HAV, respectively. For the Weibull model, estimated activation energies for FCV-F9, MNV-1, and HAV were 214 ± 28, 242 ± 36, and 154 ± 19 kJ/mole, respectively, while the calculated activation energies for the first-order model were 181 ± 16, 196 ± 5, and 167 ± 9 kJ/mole, respectively. Precise information on the thermal inactivation of HNoV surrogates and HAV in turkey deli meat was generated. This provided calculations of parameters for more-reliable thermal processes to inactivate viruses in contaminated presliced ready-to-eat deli meats and thus to reduce the risk of foodborne illness outbreaks.

Mathematical model for viral depuration kinetics in shellfish: an useful tool to estimate the risk for the consumers.

Enteric virus depuration from shellfish is a complex biological process that may be influenced by biological properties of the mollusc and/or virus species. On the basis of previous experimental data, a mathematical model was developed to characterize the kinetics of viral elimination during the depuration process. The experimental data consisted on twenty depuration trials, each with 60 kg of Manila clams (Venerupis philippinarum) and mediterranean mussels (Mytilus galloprovincialis) previously subjected to bioaccumulation with HAV or MNV-1 (as a surrogate for human norovirus), that were performed in an experimental depuration system during 7 days. It was observed that although viral loads decay along depuration, a residual viral load remains at the end of the process suggesting a decomposition of viral load in a diluted load (susceptible of depuration) and a non-diluted load (unavailable to depurate). The model yielded a general equation, which can predict the viral load at any depuration time knowing the specific filtration rate, dependent on the bivalve species, and specific viral properties. The mathematical model can be combined with quantitative risk assessment calculations to determine the safety of the depurated shellfish, which can be very helpful not only for shellfish producers but also to public health authorities.

Structural basis for host membrane remodeling induced by protein 2B of hepatitis A virus.

UNLABELLED: The complexity of viral RNA synthesis and the numerous participating factors require a mechanism to topologically coordinate and concentrate these multiple viral and cellular components, ensuring a concerted function. Similarly to all other positive-strand RNA viruses, picornaviruses induce rearrangements of host intracellular membranes to create structures that act as functional scaffolds for genome replication. The membrane-targeting proteins 2B and 2C, their precursor 2BC, and protein 3A appear to be primarily involved in membrane remodeling. Little is known about the structure of these proteins and the mechanisms by which they induce massive membrane remodeling. Here we report the crystal structure of the soluble region of hepatitis A virus (HAV) protein 2B, consisting of two domains: a C-terminal helical bundle preceded by an N-terminally curved five-stranded antiparallel ß-sheet that displays striking structural similarity to the ß-barrel domain of enteroviral 2A proteins. Moreover, the helicoidal arrangement of the protein molecules in the crystal provides a model for 2B-induced host membrane remodeling during HAV infection. IMPORTANCE: No structural information is currently available for the 2B protein of any picornavirus despite it being involved in a critical process in viral factory formation: the rearrangement of host intracellular membranes. Here we present the structure of the soluble domain of the 2B protein of hepatitis A virus (HAV). Its arrangement, both in crystals and in solution under physiological conditions, can help to understand its function and sheds some light on the membrane rearrangement process, a putative target of future antiviral drugs. Moreover, this first structure of a picornaviral 2B protein also unveils a closer evolutionary relationship between the hepatovirus and enterovirus genera within the Picornaviridae family.

Hepatitis A virus and the origins of picornaviruses.

Hepatitis A virus (HAV) remains enigmatic, despite 1.4 million cases worldwide annually. It differs radically from other picornaviruses, existing in an enveloped form and being unusually stable, both genetically and physically, but has proved difficult to study. Here we report high-resolution X-ray structures for the mature virus and the empty particle. The structures of the two particles are indistinguishable, apart from some disorder on the inside of the empty particle. The full virus contains the small viral protein VP4, whereas the empty particle harbours only the uncleaved precursor, VP0. The smooth particle surface is devoid of depressions that might correspond to receptor-binding sites. Peptide scanning data extend the previously reported VP3 antigenic site, while structure-based predictions suggest further epitopes. HAV contains no pocket factor and can withstand remarkably high temperature and low pH, and empty particles are even more robust than full particles. The virus probably uncoats via a novel mechanism, being assembled differently to other picornaviruses. It utilizes a VP2 'domain swap' characteristic of insect picorna-like viruses, and structure-based phylogenetic analysis places HAV between typical picornaviruses and the insect viruses. The enigmatic properties of HAV may reflect its position as a link between 'modern' picornaviruses and the more 'primitive' precursor insect viruses; for instance, HAV retains the ability to move from cell-to-cell by transcytosis.

Synergistic effect of high hydrostatic pressure (HHP) and marination treatment on the inactivation of hepatitis a virus in mussels (Mytilus galloprovincialis).

Consumption of raw or insufficiently cooked mussels contaminated with hepatitis A virus (HAV) is a major cause of infection to humans. The origin of mussels commonly used for the preparation of marinated seafood salads is often unknown, since different producers worldwide undergo a precooking treatment at the original collection site with methods and parameters not always indicated. These treatments could be insufficient for the inactivation of HAV, which is characterized by a high temperature resistance. Both high hydrostatic pressure (HHP) and marinade treatments have been shown to affect HAV vitality. In this study, two treatments (HHP and marinating) were combined in order to assess a potential synergistic effect on the virus vitality. A kinetic test was conducted by subjecting the experimentally-contaminated mussels (HAV titre: 10(6)/ml TCID50) to marinating, and to different HHP treatment (4,000; 5,000; and 6,000 bar for 1, 5, and 9 min). Virus post-treatment vitality was assessed by its ability to grow on cell cultures and by quantitative real-time RT-PCR to evaluate virus resistance under such conditions. Marinating treatment alone (final pH 4.3, and NaCl 2 %) did not inactivate the virus. On the other hand, the use of HHP treatment alone on non-marinated HAV-contaminated mussels was effective only above 5,000 bar for 5 min. The results of the present study elucidate the synergistic effect of a combination between marination and HHP treatments on the inactivation of the virus.

The VP4 peptide of hepatitis A virus ruptures membranes through formation of discrete pores.

UNLABELLED: Membrane-active peptides, components of capsid structural proteins, assist viruses in overcoming the host membrane barrier in the initial stages of infection. Several such peptides have been identified, and their roles in membrane fusion or disruption have been characterized through biophysical studies. In several members of the Picornaviridae family, the role of the VP4 structural peptide in cellular-membrane penetration is well established. However, there is not much information on the membrane-penetrating capsid components of hepatitis A virus (HAV), an unusual member of this family. The VP4 peptide of HAV differs from its analogues in other picornaviruses in being significantly shorter in length and in lacking a signal for myristoylation, thought to be a critical requisite for VP4-mediated membrane penetration. Here we report, for the first time, that the atypical VP4 in HAV contains significant membrane-penetrating activity. Using a combination of biophysical assays and molecular dynamics simulation studies, we show that VP4 integrates into membrane vesicles through its N-terminal region to finally form discrete pores of 5- to 9-nm diameter, which induces leakage in the vesicles without altering their overall size or shape. We further demonstrate that the membrane activity of VP4 is specific toward vesicles mimicking the lipid content of late endosomes at acidic pH. Taken together, our data indicate that VP4 might be essential for the penetration of host endosomal membranes and release of the viral genome during HAV entry. IMPORTANCE: Hepatitis A virus causes acute hepatitis in humans through the fecal-oral route and is particularly prevalent in underdeveloped regions with poor hygienic conditions. Although a vaccine for HAV exists, its high cost makes it unsuitable for universal application in developing countries. Studies on host-virus interaction for HAV have been hampered due to a lack of starting material, since the virus is extremely slow growing in culture. Among the unknown aspects of the HAV life cycle is its manner of host membrane penetration, which is one of the most important initial steps in viral infection. Here, we present data to suggest that a small peptide, VP4, a component of the HAV structural polyprotein, might be essential in helping the viral genome cross cell membranes during entry. It is hoped that this work might help in elucidating the manner of initial host cell interaction by HAV.

A comparison of the thermal inactivation kinetics of human norovirus surrogates and hepatitis A virus in buffered cell culture medium.

Human noroviruses and hepatitis A virus (HAV) are considered as epidemiologically significant causes of foodborne disease. Therefore, studies are needed to bridge existing data gaps and determine appropriate parameters for thermal inactivation of human noroviruses and HAV. The objectives of this research were to compare the thermal inactivation kinetics of human norovirus surrogates (murine norovirus (MNV-1), and feline calicivirus (FCV-F9)) and HAV in buffered medium (2-ml vials), compare first-order and Weibull models to describe the data, calculate Arrhenius activation energy for each model, and evaluate model efficiency using selected statistical criteria. The D-values calculated from the first-order model (50-72 °C) ranged from 0.21-19.75 min for FCV-F9, 0.25-36.28 min for MNV-1, and 0.88-56.22 min for HAV. Using the Weibull model, the tD = 1 (time to destroy 1 log) for FCV-F9, MNV-1 and HAV at the same temperatures ranged from 0.10-13.27, 0.09-26.78, and 1.03-39.91 min, respectively. The z-values for FCV-F9, MNV-1, and HAV were 9.66 °C, 9.16 °C, and 14.50 °C, respectively, using the Weibull model. For the first order model, z-values were 9.36 °C, 9.32 °C, and 12.49 °C for FCV-F9, MNV-1, and HAV, respectively. For the Weibull model, estimated activation energies for FCV-F9, MNV-1, and HAV were 225, 278, and 182 kJ/mol, respectively, while the calculated activation energies for the first order model were 195, 202, and 171 kJ/mol, respectively. Knowledge of the thermal inactivation kinetics of norovirus surrogates and HAV will allow the development of processes that produce safer food products and improve consumer safety.

Determination of thermal inactivation kinetics of hepatitis A virus in blue mussel (Mytilus edulis) homogenate.

Hepatitis A virus (HAV) is a food-borne enteric virus responsible for outbreaks of hepatitis associated with shellfish consumption. The objectives of this study were to determine the thermal inactivation behavior of HAV in blue mussels, to compare the first-order and Weibull models to describe the data, to calculate Arrhenius activation energy for each model, and to evaluate model efficiency by using selected statistical criteria. The times required to reduce the population by 1 log cycle (D-values) calculated from the first-order model (50 to 72°C) ranged from 1.07 to 54.17 min for HAV. Using the Weibull model, the times required to destroy 1 log unit (tD = 1) of HAV at the same temperatures were 1.57 to 37.91 min. At 72°C, the treatment times required to achieve a 6-log reduction were 7.49 min for the first-order model and 8.47 min for the Weibull model. The z-values (changes in temperature required for a 90% change in the log D-values) calculated for HAV were 15.88 ± 3.97°C (R(2), 0.94) with the Weibull model and 12.97 ± 0.59°C (R(2), 0.93) with the first-order model. The calculated activation energies for the first-order model and the Weibull model were 165 and 153 kJ/mol, respectively. The results revealed that the Weibull model was more appropriate for representing the thermal inactivation behavior of HAV in blue mussels. Correct understanding of the thermal inactivation behavior of HAV could allow precise determination of the thermal process conditions to prevent food-borne viral outbreaks associated with the consumption of contaminated mussels.

Depuration kinetics of hepatitis A virus in clams.

The efficacy and dynamic of depuration for the removal of hepatitis A virus (HAV) contamination were evaluated under experimental conditions using Manila clams previously subjected to bioaccumulation with this virus. Five independent trials were assayed in a closed experimental system with a total volume of approximately 1750 l, using clam batches of 60 Kg. The reverse transcriptase-real time PCR (RT-qPCR) technique was utilized for viral quantification. Infectivity assays were conducted at the end of depuration. Although the final viral loads in shellfish after 7 days remained relatively high and still infectious, an average reduction in HAV levels of 1.44 log units (approx. 93.1%) was observed. This reduction showed a two-phase removal kinetic, with an initial rapid reduction of viruses during the first 72 h of depuration, with a 0.6 log units (69%) of average decrease in HAV RNA copies/g digestive tissue, and a subsequent stabilization with a slower depuration rate in the remaining days.

A pathogenic picornavirus acquires an envelope by hijacking cellular membranes.

Animal viruses are broadly categorized structurally by the presence or absence of an envelope composed of a lipid-bilayer membrane, attributes that profoundly affect stability, transmission and immune recognition. Among those lacking an envelope, the Picornaviridae are a large and diverse family of positive-strand RNA viruses that includes hepatitis A virus (HAV), an ancient human pathogen that remains a common cause of enterically transmitted hepatitis. HAV infects in a stealth-like manner and replicates efficiently in the liver. Virus-specific antibodies appear only after 3-4 weeks of infection, and typically herald its resolution. Although unexplained mechanistically, both anti-HAV antibody and inactivated whole-virus vaccines prevent disease when administered as late as 2 weeks after exposure, when virus replication is well established in the liver. Here we show that HAV released from cells is cloaked in host-derived membranes, thereby protecting the virion from antibody-mediated neutralization. These enveloped viruses ('eHAV') resemble exosomes, small vesicles that are increasingly recognized to be important in intercellular communications. They are fully infectious, sensitive to extraction with chloroform, and circulate in the blood of infected humans. Their biogenesis is dependent on host proteins associated with endosomal-sorting complexes required for transport (ESCRT), namely VPS4B and ALIX. Whereas the hijacking of membranes by HAV facilitates escape from neutralizing antibodies and probably promotes virus spread within the liver, anti-capsid antibodies restrict replication after infection with eHAV, suggesting a possible explanation for prophylaxis after exposure. Membrane hijacking by HAV blurs the classic distinction between 'enveloped' and 'non-enveloped' viruses and has broad implications for mechanisms of viral egress from infected cells as well as host immune responses.