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.

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

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

Biochemical and structural characterization of hepatitis A virus 2C reveals an unusual ribonuclease activity on single-stranded RNA.

The HAV nonstructural protein 2C is essential for virus replication; however, its precise function remains elusive. Although HAV 2C shares 24-27% sequence identity with other 2Cs, key motifs are conserved. Here, we demonstrate that HAV 2C is an ATPase but lacking helicase activity. We identified an ATPase-independent nuclease activity of HAV 2C with a preference for polyuridylic single-stranded RNAs. We determined the crystal structure of an HAV 2C fragment to 2.2 Å resolution, containing an ATPase domain, a region equivalent to enterovirus 2C zinc-finger (ZFER) and a C-terminal amphipathic helix (PBD). The PBD of HAV 2C occupies a hydrophobic pocket (Pocket) in the adjacent 2C, and we show the PBD-Pocket interaction is vital for 2C functions. We identified acidic residues that are essential for the ribonuclease activity and demonstrated mutations at these sites abrogate virus replication. We built a hexameric-ring model of HAV 2C, revealing the ribonuclease-essential residues clustering around the central pore of the ring, whereas the ATPase active sites line up at the gaps between adjacent 2Cs. Finally, we show the ribonuclease activity is shared by other picornavirus 2Cs. Our findings identified a previously unfound activity of picornavirus 2C, providing novel insights into the mechanisms of virus replication.

Evaluation of Potential Anti-Hepatitis A Virus 3C Protease Inhibitors Using Molecular Docking.

Hepatitis A virus (HAV) infection is a major cause of acute hepatitis worldwide and occasionally causes acute liver failure and can lead to death in the absence of liver transplantation. Although HAV vaccination is available, the prevalence of HAV vaccination is not adequate in some countries. Additionally, the improvements in public health reduced our immunity to HAV infection. These situations motivated us to develop potentially new anti-HAV therapeutic options. We carried out the in silico screening of anti-HAV compounds targeting the 3C protease enzyme using the Schrodinger Modeling software from the antiviral library of 25,000 compounds to evaluate anti-HAV 3C protease inhibitors. Additionally, in vitro studies were introduced to examine the inhibitory effects of HAV subgenomic replicon replication and HAV HA11-1299 genotype IIIA replication in hepatoma cell lines using luciferase assays and real-time RT-PCR. In silico studies enabled us to identify five lead candidates with optimal binding interactions in the active site of the target HAV 3C protease using the Schrodinger Glide program. In vitro studies substantiated our hypothesis from in silico findings. One of our lead compounds, Z10325150, showed 47% inhibitory effects on HAV genotype IB subgenomic replicon replication and 36% inhibitory effects on HAV genotype IIIA HA11-1299 replication in human hepatoma cell lines, with no cytotoxic effects at concentrations of 100 µg/mL. The effects of the combination therapy of Z10325150 and RNA-dependent RNA polymerase inhibitor, favipiravir on HAV genotype IB HM175 subgenomic replicon replication and HAV genotype IIIA HA11-1299 replication showed 64% and 48% inhibitory effects of HAV subgenomic replicon and HAV replication, respectively. We identified the HAV 3C protease inhibitor Z10325150 through in silico screening and confirmed the HAV replication inhibitory activity in human hepatocytes. Z10325150 may offer the potential for a useful HAV inhibitor in severe hepatitis A.

Oligomers of hepatitis A virus (HAV) capsid protein VP1 generated in a heterologous expression system.

BACKGROUND: The quasi-enveloped picornavirus, Hepatitis A Virus (HAV), causes acute hepatitis in humans and infects approximately 1.5 million individuals a year, which does not include the asymptomatically infected population. Several severe outbreaks in developing nations in recent years have highlighted the reduction in HAV endemicity, which increases the risk of infections in the vulnerable population. The current HAV vaccines are based on growing wildtype or attenuated virus in cell culture, which raises the cost of production. For generation of cheaper, subunit vaccines or strategies for antibody-based diagnostics, production of viral structural proteins in recombinant form in easily accessible expression systems is a priority. RESULTS: We attempted several strategies for recombinant production of one of the major capsid proteins VP1, from HAV, in the E. coli expression system. Several efforts resulted in the formation of soluble aggregates or tight association of VP1 with the bacterial chaperone GroEL. Correctly folded VP1 was eventually generated in a discrete oligomeric form upon purification of the protein from inclusion bodies and refolding. The oligomers resemble oligomers of capsid proteins from other picornaviruses and appear to have the correct secondary and antigenic surface structure. CONCLUSIONS: VP1 oligomers generated in the bacterial expression system can be utilized for understanding the molecular pathway of HAV capsid assembly and may also have potential biomedical usages in prevention and diagnostics of HAV infections.

Favipiravir Inhibits Hepatitis A Virus Infection in Human Hepatocytes.

Hepatitis A virus (HAV) is a causative agent of acute hepatitis and can occasionally induce acute liver failure. However, specific potent anti-HAV drug is not available on the market currently. Thus, we investigated several novel therapeutic drugs through a drug repositioning approach, targeting ribonucleic acid (RNA)-dependent RNA polymerase and RNA-dependent deoxyribonucleic acid polymerase. In the present study, we examined the anti-HAV activity of 18 drugs by measuring the HAV subgenomic replicon and HAV HA11-1299 genotype IIIA replication in human hepatoma cell lines, using a reporter assay and real-time reverse transcription polymerase chain reaction, respectively. Mutagenesis of the HAV 5' untranslated region was also examined by next-generation sequencing. These specific parameters were explored because lethal mutagenesis has emerged as a novel potential therapeutic approach to treat RNA virus infections. Favipiravir inhibited HAV replication in both Huh7 and PLC/PRF/5 cells, although ribavirin inhibited HAV replication in only Huh7 cells. Next-generation sequencing demonstrated that favipiravir could introduce nucleotide mutations into the HAV genome more than ribavirin. In conclusion, favipiravir could introduce nucleotide mutations into the HAV genome and work as an antiviral against HAV infection. Provided that further in vivo experiments confirm its efficacy, favipiravir would be useful for the treatment of severe HAV infection.

Efficient capturing and sensitive detection of hepatitis A virus from solid foods (green onion, strawberry, and mussel) using protamine-coated iron oxide (Fe3O4) magnetic nanoparticles and real-time RT-PCR.

Hepatitis A virus (HAV) continues to be a public health concern and has caused large foodborne outbreaks and economic losses worldwide. Rapid detection of HAV in foods can help to confirm the source of outbreaks in a timely manner and prevent more people getting infected. In order to efficiently detect HAV at low levels of contamination in foods, rapid and easy-to-use techniques are required to separate and concentrate viral particles to a small volume. In the current study, HAV particles were eluted from green onion, strawberry, and mussel using glycine buffer (0.05 M glycine, 0.14 M NaCl, 0.2% (v/v) Tween 20, pH 9.0) and suspended viral particles were captured using protamine-coated magnetic nanoparticles (PMNPs). This process caused a selective concentration of the viral particles, which could be followed by quantitative real-time RT-PCR analysis. Results showed that pH, NaCl concentration, and PMNP amount used for the capturing had significant effects on the recovery efficiency of HAV (P < 0.05). The highest recovery rate was obtained at pH 9.0, 0.14 M NaCl, and 50 µL of PMNPs. The optimized PMNP capturing method enabled the rapid capture and concentration of HAV. A sensitive real-time RT-PCR test was developed with detection limits of 8.3 × 100 PFU/15 g, 8.3 × 101 PFU/50 g, and 8.3 × 100 PFU/5 g of HAV in green onion, strawberry, and mussel, respectively. In conclusion, the PMNP method is rapid and convenient in capturing HAV from complex solid food samples and can generate concentrated HAV sample solutions suitable for high-sensitivity real time RT-PCR detection of the virus.

Transfusion-Transmitted Hepatitis A Virus, France, 2018.

We report a transfusion-transmitted hepatitis A virus infection in an immunocompromised patient in France, detected shortly after a transfusion of pathogen-reduced pooled platelets. This case raises questions about the efficacy of donor screening methods. Additional safety measures, such as routine donation screening, should be considered.

Hepatitis A and E among immigrants and refugees in Central Brazil

ABSTRACT OBJECTIVE To estimate the prevalence of hepatitis A virus (HAV) and hepatitis E virus (HEV) among immigrants and refugees in Goiás, Central Brazil. METHODS Overall, 355 individuals were interviewed, and blood samples were tested for anti-HAV and anti-HEV IgG. Anti-HEV-positive samples were similarly tested for HEV RNA. RESULTS All participants were from Latin American countries, most of whom, young adult males. The overall anti-HAV IgG prevalence was 87.4% (95%CI: 83.5-90.4), of whom 94.9%, 75.6%, and 60% were from Haiti, Venezuela, and other Latin American countries, respectively (p < 0.001). Age above 19 years and more than 36 months residing in Brazil were associated with a higher prevalence of previous HAV and HEV infection, respectively. Of the children eligible for HAV vaccination according to the National Immunization Program, only eight (44%) had been vaccinated. The overall anti-HEV IgG prevalence was 6.5% (95%CI: 4.4-9.5). All anti-HEV IgG-positive individuals were Haitians, including a child born in Brazil. HEV RNA was detected in two of the anti-HEV IgG-positive samples. CONCLUSION The survey detected a high prevalence of anti-HAV and anti-HEV IgG among immigrants and refugees, and active HEV infection among some Haitian participants. Prevention measures are urgently required to interrupt enteric virus transmission in this emergent and vulnerable population.

Additive Effects of Zinc Chloride on the Suppression of Hepatitis A Virus Replication by Interferon in Human Hepatoma Huh7 Cells.

BACKGROUND/AIM: Hepatitis A virus (HAV) infection is still one of the serious health problems worldwide, despite the existence of effective vaccines for HAV. Zinc compounds have antiviral activities against various DNA and RNA viruses. Therefore, we investigated the effects of zinc compounds on the antiviral activity of interferon against HAV. MATERIALS AND METHODS: The effects of zinc compounds with or without interferon on HAV genotype IIIA HA11-1299 replication were examined in human hepatoma Huh7 cells. Cell viability was examined by the MTS assay. Inflammasome associated gene expression was examined by real-time reverse transcription-polymerase chain reaction. RESULTS: Both zinc sulfate and zinc chloride had an inhibitory effect on HAV replication. Zinc sulfate tended to enhance while zinc chloride significantly enhanced the anti-HAV effect induced by interferon-alpha-2a. Zinc chloride significantly up-regulated mitogen-activated protein kinase 12 (MAPK12) and down-regulated 6 related genes [baculoviral IAP repeat containing 3 (BIRC3), interleukin 1 beta (IL1B), proline-serine-threonine phosphatase interacting protein 1 (PSTPIP1), prostaglandin-endoperoxide synthase 2 (PTGS2), PYD and CARD domain containing (PYCARD), and tumor necrosis factor (TNF)]. CONCLUSION: Zinc chloride inhibits HAV replication and has additive effects on the anti-HAV activities of interferon.

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.

Gangliosides are essential endosomal receptors for quasi-enveloped and naked hepatitis A virus.

The Picornaviridae are a diverse family of positive-strand RNA viruses that includes numerous human and veterinary pathogens1. Among these, hepatitis A virus (HAV), a common cause of acute hepatitis in humans, is unique in that it is hepatotropic and is released from hepatocytes without lysis in small vesicles that resemble exosomes2,3. These quasi-enveloped virions are infectious and are the only form of virus that can be detected in the blood during acute infection2. By contrast, non-enveloped naked virions are shed in faeces and stripped of membranes by bile salts during passage through the bile ducts to the gut4. How these two distinct types of infectious hepatoviruses enter cells to initiate infection is unclear. Here, we describe a genome-wide forward screen that shows that glucosylceramide synthase and other components of the ganglioside synthetic pathway are crucial host factors that are required for cellular entry by hepatoviruses. We show that gangliosides-preferentially disialogangliosides-function as essential endolysosome receptors that are required for infection by both naked and quasi-enveloped virions. In the absence of gangliosides, both virion types are efficiently internalized through endocytosis, but capsids fail to uncoat and accumulate within LAMP1+ endolysosomes. Gangliosides relieve this block, binding to the capsid at low pH and facilitating a late step in entry involving uncoating and delivery of the RNA genome to the cytoplasm. These results reveal an atypical cellular entry pathway for hepatoviruses that is unique among picornaviruses.

Persistence of murine norovirus, bovine rotavirus, and hepatitis A virus on stainless steel surfaces, in spring water, and on blueberries.

The viability of murine norovirus (MNV-1), bovine rotavirus (boRV), and hepatitis A virus (HAV) was evaluated at 21 °C, 4 °C, and -20 °C on stainless steel surfaces, in bottled water, and on blueberries for up to 21 days. After 14 days of incubation at 21 °C on stainless steel, a viability loss >4 log for MNV-1, >8 log for boRV, and >1 log for HAV was observed. Losses were observed for MNV-1 (>1 log) and HAV (>2 log) incubated in water at 21 °C for 21 days. No significant loss was detected for MNV-1 and HAV at 4 °C and -20 °C and for boRV at 21 °C, 4 °C, and -20 °C. On blueberries incubated at 4 °C and -20 °C, they all maintained their infectivity. After 7 days at 21 °C, a loss >2 log, a loss of 3 log, and no loss were observed for boRV, MNV-1, and HAV, respectively. After RNase pretreatment, the detection of extracted RNA from infectious and noninfectious samples suggested the protection of RNA inside the capsid. Even though they all are enteric viruses, their persistence varied with temperature and the nature of the commodity. It is therefore important to use more than one viral surrogate, during inactivation treatments or implementation of control measures.

Hepatitis A outbreak affecting men who have sex with men (MSM) in central Argentina, occurred in July 2017-April 2018, later than the European outbreak.

BACKGROUND: During June-2016-May-2017, several outbreaks of HA were recorded in Europe, especially described in MSM. In our area since July-2017, an increase of hepatitis A (HA) notification was reported. OBJECTIVE: In order to understand the unusual increase of cases occurred in the central region of Argentina, the aim of this study was to describe, characterize and contextualize epidemiologically the HA outbreak occurred this area, until April2018. STUDY DESIGN: HA cases (positive anti-HAV IgM) obtained from the calendar week 29/2017 in which the first case of MSM was recognized were included in our study. HAV RNA detection and molecular characterization was performed from serum samples and/or stool by RT - PCR of VP1/2A genomic region (360bp). RESULTS: Of the 32 cases notified, 87.5% of them were unvaccinated men and 69.6% were MSM (mean age 31.9 years). All MSM associated HAV sequences were genotyped as IA, and clustered with the VRD 521-2016 strain, responsible of causing outbreaks mostly in MSM in Europe since mid-2016. CONCLUSION: As a consequence of the implementation of immunization in children, and the improvement in socio-economic, hygienic and sanitation factors, young adults are becoming increasingly susceptible to HAV infections. Here we add evidence in South America to the HA outbreaks described worldwide among young MSM, demonstrating the need to reinforce official policy of vaccination, in this group and adjust epidemiological surveillance, catch-up vaccination for adolescents, young adults and immunosuppressed patients.

Hepatitis A-2017 an unusual year in Scotland.

BACKGROUND: The number of cases of acute hepatitis A reported in Scotland each year is small, and the majority of cases have been associated with travel to endemic regions. However, in early 2017, in the midst of ongoing outbreaks of hepatitis A among MSM in Europe, there was a sharp rise in the number of cases reported to Health Protection Scotland. OBJECTIVES: The initial aim of this study was to investigate the reason for the observed increase in cases of hepatitis A at the start of 2017. As cases continued for the remainder of the year, these cases were typed to determine whether these cases were linked to each other, or other outbreaks. STUDY DESIGN: The study population consisted of 42 hepatitis A infected patients with no obvious source of infection. The patient samples were collected between January and December 2017. The VP1/2 A region was amplified and sequenced. RESULTS: The majority of samples typed as genotype 1 A (n = 17) or genotype 1B (n = 15). Within genotype 1 A, fifteen samples had strains (VRD_521_2016 or RIVM_HAV16_090) associated with ongoing outbreaks of hepatitis A in MSM in Europe. Within genotype 1B, there were four clusters of infections, with identical cases in geographically distinct regions with no identified epidemiological link. CONCLUSIONS: Molecular typing proved useful, as it allowed public health to identify clusters, establish links with other outbreaks and compare Scottish strains with those reported elsewhere.

Mineral Waste Containing High Levels of Iron from an Environmental Disaster (Bento Rodrigues, Mariana, Brazil) is Associated with Higher Titers of Enteric Viruses.

Although the effects of heavy metals on the behavior, including infectivity, of bacteria have been studied, little information is available about their effects on enteric viruses. We report an investigation of effects on the biosynthesis of human adenoviruses (HAdV) and hepatitis A (HAV) of waters contaminated with mineral waste following an environmental disaster in Mariana City, Minas Gerais State, Brazil. The study area was affected on November 5, 2015, by 60 million m3 of mud (containing very high concentrations of iron salts) from a mining reservoir (Fundão), reaching the Gualaxo do Norte River (sites evaluated in this study), the "Rio Doce" River and finally the Atlantic Ocean. We found substantial counts of infectious HAdV and HAV (by qPCR) in all sampled sites from Gualaxo do Norte River, indicating poor basic sanitation in this area. The effects of iron on viral infection processes were evaluated using HAdV-2 and HAV-175, as DNA and RNA enteric virus models, respectively, propagated in the laboratory and exposed to this contaminated water. Experiments in field and laboratory scales found that the numbers of plaque forming units (PFU) of HAdV and HAV were significantly higher in contaminated water with high iron concentrations than in waters with low iron concentration (< 20 µg/L of iron). These findings indicate that iron can potentiate enteric virus infectivity, posing a potential risk to human and animal health, particularly during pollution disasters such as that described here in Mariana, Brazil.

Detection of Subgenotype IA and IIIA Hepatitis A Viruses in Rivers Flowing through Metro Manila, the Philippines.

Hepatitis A virus (HAV) is a common infectious etiology of acute hepatitis worldwide. The Philippines remains highly endemic for hepatitis A, but there is still a lack of information about HAV in the country. To evaluate the HAV contamination in environmental water in the Philippines, we conducted the detection and genetic analyses of HAV RNA in samples from river water. Twelve water samples were collected at 6 sampling sites of 3 rivers in Metro Manila, in both the dry and wet seasons in 2012 and 2013. The HAV RNA was detected in all the 6 samples collected in the dry season, and in one sample from the wet season. Phylogenetic analysis confirmed that the HAV strains detected in the river water included multiple sequences belonging to subgenotypes IA and IIIA. This indicates that at least 2 genotypes of the HAV strains are circulating in the environment in the Philippines, posing a risk of HAV infection to not only residents, but also tourists, especially in the dry season.

Detection and quantification of human adenovirus (HAdV), JC polyomavirus (JCPyV) and hepatitis A virus (HAV) in recreational waters of Niterói, Rio de Janeiro, Brazil.

This study evaluated the impact of sewage discharge in recreational coastal marine environments of Niteroi, Rio de Janeiro, Brazil, over a six-month period by the detection of waterborne enteric viruses. Ten-liter water samples were collected in four beaches from January to July 2017. Viruses were concentrated by an organic flocculation and human adenoviruses (HAdV), polyomavirus (JCPyV), and Hepatitis A virus (HAV) detected by qPCR. Forty-eight water samples were collected, being 43% positive for HAdV and 23% for JCPyV; only one sample was positive for HAV. Viruses were detected in all sampling sites, including in areas suitable for bathing according to the current bacterial standards. The results herein provide an overview of the viral contamination of beaches used for recreational purposes. The viral presence in the sampled areas indicates the need for more rigid effluent discharge controls in these areas, as sewage represents a possible transmission risk for waterborne viral diseases.

A Novel Marsupial Hepatitis A Virus Corroborates Complex Evolutionary Patterns Shaping the Genus Hepatovirus.

The discovery of highly diverse nonprimate hepatoviruses illuminated the evolutionary origins of hepatitis A virus (HAV) ancestors in mammals other than primates. Marsupials are ancient mammals that diverged from other Eutheria during the Jurassic. Viruses from marsupials may thus provide important insight into virus evolution. To investigate Hepatovirus macroevolutionary patterns, we sampled 112 opossums in northeastern Brazil. A novel marsupial HAV (MHAV) in the Brazilian common opossum (Didelphis aurita) was detected by nested reverse transcription-PCR (RT-PCR). MHAV concentration in the liver was high, at 2.5 × 109 RNA copies/g, and at least 300-fold higher than those in other solid organs, suggesting hepatotropism. Hepatovirus seroprevalence in D. aurita was 26.6% as determined using an enzyme-linked immunosorbent assay (ELISA). Endpoint titers in confirmatory immunofluorescence assays were high, and marsupial antibodies colocalized with anti-HAV control sera, suggesting specificity of serological detection and considerable antigenic relatedness between HAV and MHAV. MHAV showed all genomic hallmarks defining hepatoviruses, including late-domain motifs likely involved in quasi-envelope acquisition, a predicted C-terminal pX extension of VP1, strong avoidance of CpG dinucleotides, and a type 3 internal ribosomal entry site. Translated polyprotein gene sequence distances of at least 23.7% from other hepatoviruses suggested that MHAV represents a novel Hepatovirus species. Conserved predicted cleavage sites suggested similarities in polyprotein processing between HAV and MHAV. MHAV was nested within rodent hepatoviruses in phylogenetic reconstructions, suggesting an ancestral hepatovirus host switch from rodents into marsupials. Cophylogenetic reconciliations of host and hepatovirus phylogenies confirmed that host-independent macroevolutionary patterns shaped the phylogenetic relationships of extant hepatoviruses. Although marsupials are synanthropic and consumed as wild game in Brazil, HAV community protective immunity may limit the zoonotic potential of MHAV.IMPORTANCE Hepatitis A virus (HAV) is a ubiquitous cause of acute hepatitis in humans. Recent findings revealed the evolutionary origins of HAV and the genus Hepatovirus defined by HAV in mammals other than primates in general and in small mammals in particular. The factors shaping the genealogy of extant hepatoviruses are unclear. We sampled marsupials, one of the most ancient mammalian lineages, and identified a novel marsupial HAV (MHAV). The novel MHAV shared specific features with HAV, including hepatotropism, antigenicity, genome structure, and a common ancestor in phylogenetic reconstructions. Coevolutionary analyses revealed that host-independent evolutionary patterns contributed most to the current phylogeny of hepatoviruses and that MHAV was the most drastic example of a cross-order host switch of any hepatovirus observed so far. The divergence of marsupials from other mammals offers unique opportunities to investigate HAV species barriers and whether mechanisms of HAV immune control are evolutionarily conserved.

Duck hepatitis A virus structural proteins expressed in insect cells self-assemble into virus-like particles with strong immunogenicity in ducklings.

Duck hepatitis A virus (DHAV), a non-enveloped ssRNA virus, can cause a highly contagious disease in young ducklings. The three capsid proteins of VP0, VP1 and VP3 are translated within a single large open reading frame (ORF) and hydrolyzed by protease 3CD. However, little is known on whether the recombinant viral structural proteins (VPs) expressed in insect cells could spontaneously assemble into virus-like particles (VLPs) and whether these VLPs could induce protective immunity in young ducklings. To address these issues, the structural polyprotein precursor gene P1 and the protease gene 3CD were amplified by PCR, and the recombinant proteins were expressed in insect cells using a baculovirus expression system for the characterization of their structures and immunogenicity. The recombinant proteins expressed in Sf9 cells were detected by indirect immunofluorescence assay and Western blot analysis. Electron microscopy showed that the recombinant proteins spontaneously assembled into VLPs in insect cells. Western blot analysis of the purified VLPs revealed that the VLPs were composed with the three structural proteins. In addition, vaccination with the VLPs induced high humoral immune response and provided strong protection. Therefore, our findings may provide a framework for development of new vaccines for the prevention of duck viral hepatitis.