Genetic substructure and host-specific natural selection trend across vaccine-candidate ORF-2 capsid protein of hepatitis-E virus.

Hepatitis E virus is a primary cause of acute hepatitis worldwide. The present study attempts to assess the genetic variability and evolutionary divergence among HEV genotypes. A vaccine promising capsid-protein coding ORF-2 gene sequences of HEV was evaluated using phylogenetics, model-based population genetic methods and principal component analysis. The analyses unveiled nine distinct clusters as subpopulations for six HEV genotypes. HEV-3 genotype samples stratified into four different subgroups, while HEV-4 stratified into three additional subclusters. Rabbit-infectious HEV-3ra samples constitute a distinct cluster. Pairwise analysis identified marked genetic distinction of HEV-4c and HEV-4i subgenotypes compared to other genotypes. Numerous admixed, inter and intragenotype recombinant strains were detected. The MEME method identified several ORF-2 codon sites under positive selection. Some selection signatures lead to amino acid substitutions within ORF-2, resulting in altered physicochemical features. Moreover, a pattern of host-specific adaptive signatures was identified among HEV genotypes. The analyses conclusively depict that recombination and episodic positive selection events have shaped the observed genetic diversity among different HEV genotypes. The significant genetic diversity and stratification of HEV-3 and HEV-4 genotypes into subgroups, as identified in the current study, are noteworthy and may have implications for the efficacy of anti-HEV vaccines.

Development of a HiBiT-tagged reporter hepatitis E virus and its utility as an antiviral drug screening platform.

Previously, we developed an infectious hepatitis E virus (HEV) harboring the nanoKAZ gene in the hypervariable region of the open reading frame 1 (ORF1) of the HEV3b (JE03-1760F/P10) genome and demonstrated the usefulness for screening anti-HEV drugs that inhibit the early infection process. In the present study, we constructed another reporter HEV (HEV3b-HiBiT) by placing a minimized HiBiT tag derived from NanoLuc luciferase at the 3'-end of the viral capsid (ORF2) coding sequence. It replicated efficiently in PLC/PRF/5 cells, produced membrane-associated particles identical to those of the parental virus, and was genetically stable and infectious. The HiBiT tag was fused to both secreted ORF2s (ORF2s-HiBiT) and ORF2c capsid protein (ORF2c-HiBiT). The ORF2c-HiBiT formed membrane-associated HEV particles (eHEV3b-HiBiT). By treating these particles with digitonin, we demonstrated that the HiBiT tag was expressed on the surface of capsid and was present inside the lipid membrane. To simplify the measurement of luciferase activity and provide a more convenient screening platform, we constructed an ORF2s-defective mutant (HEV3b-HiBiT/ΔORF2s) in which the secreted ORF2s are suppressed. We used this system to evaluate the effects of introducing small interfering RNAs and treatment with an inhibitor or accelerator of exosomal release on HEV egress and demonstrated that the effects on virus release can readily be analyzed. Therefore, HEV3b-HiBiT and HEV3b-HiBiT/ΔORF2s reporters may be useful for investigating the virus life cycle and can serve as a more convenient screening platform to search for candidate drugs targeting the late stage of HEV infection such as particle formation and release. IMPORTANCE The construction of recombinant infectious viruses harboring a stable luminescence reporter gene is essential for investigations of the viral life cycle, such as viral replication and pathogenesis, and the development of novel antiviral drugs. However, it is difficult to maintain the stability of a large foreign gene inserted into the viral genome. In the present study, we successfully generated a recombinant HEV harboring the 11-amino acid HiBiT tag in the ORF2 coding region and demonstrated the infectivity, efficient virus growth, particle morphology, and genetic stability, suggesting that this recombinant HEV is useful for in vitro assays. Furthermore, this system can serve as a more convenient screening platform for anti-HEV drugs. Thus, an infectious recombinant HEV is a powerful approach not only for elucidating the molecular mechanisms of the viral life cycle but also for the screening and development of novel antiviral agents.

The endocytic recycling compartment serves as a viral factory for hepatitis E virus.

Although hepatitis E virus (HEV) is the major leading cause of enterically transmitted viral hepatitis worldwide, many gaps remain in the understanding of the HEV lifecycle. Notably, viral factories induced by HEV have not been documented yet, and it is currently unknown whether HEV infection leads to cellular membrane modeling as many positive-strand RNA viruses. HEV genome encodes the ORF1 replicase, the ORF2 capsid protein and the ORF3 protein involved in virion egress. Previously, we demonstrated that HEV produces different ORF2 isoforms including the virion-associated ORF2i form. Here, we generated monoclonal antibodies that specifically recognize the ORF2i form and antibodies that recognize the different ORF2 isoforms. One antibody, named P1H1 and targeting the ORF2i N-terminus, recognized delipidated HEV particles from cell culture and patient sera. Importantly, AlphaFold2 modeling demonstrated that the P1H1 epitope is exposed on HEV particles. Next, antibodies were used to probe viral factories in HEV-producing/infected cells. By confocal microscopy, we identified subcellular nugget-like structures enriched in ORF1, ORF2 and ORF3 proteins and viral RNA. Electron microscopy analyses revealed an unprecedented HEV-induced membrane network containing tubular and vesicular structures. We showed that these structures are dependent on ORF2i capsid protein assembly and ORF3 expression. An extensive colocalization study of viral proteins with subcellular markers, and silencing experiments demonstrated that these structures are derived from the endocytic recycling compartment (ERC) for which Rab11 is a central player. Hence, HEV hijacks the ERC and forms a membrane network of vesicular and tubular structures that might be the hallmark of HEV infection.

Non-Assembled ORF2 Capsid Protein of Porcine Circovirus 2b Does Not Confer Protective Immunity.

Porcine Circovirus 2 (PCV2) vaccines are based on either inactivated whole virion, or recombinant ORF2 capsid protein assembled into Virus-like Particles (VLPs). No data are available about the immunizing properties of free, non-assembled capsid protein. To investigate this issue, ORF2 of a reference PCV2b strain was expressed in a Baculovirus-based expression system without assembly into VLPs. The free purified protein was formulated into an oil vaccine at three distinct Ag payloads: 10.8/3.6/1.2 micrograms/dose. Each dose was injected intramuscularly into five, 37-day old piglets, carefully matched for maternally-derived antibody. Five control piglets were injected with sterile PBS in oil adjuvant. Twenty-eight days later, all the pigs were challenged intranasally with 105.3 TCID50 of PCV2b strain DV6503. After challenge infection, all the pigs remained in good clinical conditions. The recombinant vaccine did not induce significant antibody and PCV2-specific IFN-γ responses. ELISPOT and lymphocyte proliferation data confirmed poor induction of cell-mediated immunity. In terms of PCV2 viremia, there was no significant difference between vaccinated and control animals. The histological data indicated the absence of a detectable viral load and of PCVAD lesions in both vaccinated and control animals, as well as of histiocytes and multi-nucleated giant cells. We conclude that free, non-assembled ORF2 capsid protein does not induce protective immunity.

Development and Optimization of an Enzyme Immunoassay to Detect Serum Antibodies against the Hepatitis E Virus in Pigs, Using Plant-Derived ORF2 Recombinant Protein.

Hepatitis E is an emerging global disease, mainly transmitted via the fecal-oral route in developing countries, and in a zoonotic manner in the developed world. Pigs and wild boar constitute the primary Hepatitis E virus (HEV) zoonotic reservoir. Consumption of undercooked animal meat or direct contact with infected animals is the most common source of HEV infection in European countries. The purpose of this study is to develop an enzyme immunoassay (EIA) for the detection of anti-hepatitis E virus IgG in pig serum, using plant-produced recombinant HEV-3 ORF2 as an antigenic coating protein, and also to evaluate the sensitivity and specificity of this assay. A recombinant HEV-3 ORF2 110-610_6his capsid protein, transiently expressed by pEff vector in Nicotiana benthamiana plants was used to develop an in-house HEV EIA. The plant-derived HEV-3 ORF2 110-610_6his protein proved to be antigenically similar to the HEV ORF2 capsid protein and it can self-assemble into heterogeneous particulate structures. The optimal conditions for the in-house EIA (iEIA) were determined as follows: HEV-3 ORF2 110-610_6his antigen concentration (4 µg/mL), serum dilution (1:50), 3% BSA as a blocking agent, and secondary antibody dilution (1:20 000). The iEIA developed for this study showed a sensitivity of 97.1% (95% Cl: 89.9-99.65) and a specificity of 98.6% (95% Cl: 92.5-99.96) with a Youden index of 0.9571. A comparison between our iEIA and a commercial assay (PrioCHECK™ Porcine HEV Ab ELISA Kit, ThermoFisher Scientific, MA, USA) showed 97.8% agreement with a kappa index of 0.9399. The plant-based HEV-3 ORF2 iEIA assay was able to detect anti-HEV IgG in pig serum with a very good agreement compared to the commercially available kit.

Progress in the Production of Virus-Like Particles for Vaccination against Hepatitis E Virus.

Hepatitis E virus (HEV), a pathogen that causes acute viral hepatitis, is a small icosahedral, quasi-enveloped, positive ssRNA virus. Its genome has three open reading frames (ORFs), with ORF1 and ORF3 encoding for nonstructural and regulatory proteins, respectively, while ORF2 is translated into the structural, capsid protein. ORF2 is most widely used for vaccine development in viral hepatitis. Hepatitis E virus-like particles (VLPs) are potential vaccine candidates against HEV infection. VLPs are composed of capsid subunits mimicking the natural configuration of the native virus but lack the genetic material needed for replication. As a result, VLPs are unable to replicate and cause disease, constituting safe vaccine platforms. Currently, the recombinant VLP-based vaccine Hecolin® against HEV is only licensed in China. Herein, systematic information about the expression of various HEV ORF2 sequences and their ability to form VLPs in different systems is provided.

The crystal structure of the emerging human-infecting hepatitis E virus E2s protein.

Hepatitis E virus (HEV) is a non-enveloped, globular particle that is responsible for acute hepatitis. HEV is classified into the Hepeviridae family and can be divided into four species (A-D). All HEV variants that infect humans are reported to belong to species A (HEV-A), except species C (HEV-C), which was reported to infect humans in December 2018. We determined the crystal structure of the HEV-C E2s domain at 1.8 Å resolution. It contains a classical 12-stranded ß-sandwich motif and forms dimers by hydrogen bonding, though the amino acid residues that form hydrogen bonds are quite different from the residues of HEV-A. The HEV-C E2s domain shares the common groove region with other structurally related viruses, and some subtle differences in this region may be related to host adoption or antibody binding. Antibody binding experiments and structural analysis revealed that HEV-C E2s is able to bind to the previously reported broad-spectrum antibody 8G12 but not bind to the antibody 8C11. Meanwhile, the structure analysis shows that HEV-C E2s does not have the key sites for binding to host cells as displayed by HEV-A (Genotype 1) E2s. These structural and biological findings present important implications for understanding the molecular mechanisms of host recognition and entry of HEV-C, as well as provide clues to the development of therapeutic antibodies and vaccines against HEV-C infection.

Hepatitis E virus genotype 3 and capsid protein in the blood and urine of immunocompromised patients.

OBJECTIVES: Hepatitis E virus genotype 3 (HEV3) is responsible for acute and chronic liver disease in solid organ transplant (SOT) recipients. HEV was recently found in the urine of some acutely and chronically genotype 4-infected patients. METHODS: We examined the urinary excretion of HEV3 by 24 consecutive SOT recipients at the acute phase of HEV hepatitis and characterized the excreted virus. RESULTS: Urinary HEV RNA was detected in 12 (50%) of the 24 transplanted patients diagnosed with HEV hepatitis. Urinary HEV antigen (Ag) was detected in all but one of the patients (96%). The density of RNA-containing HEV particles in urine was low (1.11-1.12 g/cm3), corresponding to lipid-associated virions. The urinary HEV RNA/Ag detected was not associated with impaired kidney function or de novo proteinuria. Finally, there was more HEV Ag in the serum at the acute phase of HEV infection in SOT recipients whose infection became chronic. CONCLUSIONS: HEV3 excreted via the urine of SOT recipients at the acute phase of HEV hepatitis has a lipid envelope. Renal function was not impaired. While urinary HEV Ag was a sensitive indicator of HEV infection, only acute phase serum HEV Ag indicated the development of a chronic infection.

Conservation and variation of the hepatitis E virus ORF2 capsid protein.

Hepatitis E virus (HEV) is one of the major pathogens causing acute viral hepatitis. The infectious particle consists of an RNA genome and capsid proteins. The 7.2 kb genome encodes three open reading frames (ORF) and ORF2 is translated into the capsid protein. The knowledge of structure and function of the ORF2 protein is essential for understanding the evolution and life cycle of HEV. However, biophysical research in this respect remains limited due to technical challenges. We have carried out a series of computational analysis on HEV ORF2. We have identified 144 conserved sites among the 660 amino acid (AA) residues. 43 models based on the previously proposed reference sequences and a cell culture adapted infectious clone were successfully built by 3D protein structure prediction and refinement. Structure alignment of domains revealed structural conservation of the S and M domains, but to a lesser extent of the P domain. Moreover, molecular docking has predicted distinct binding affinities of a monoclonal antibody towards ORF2 across different genotypes. Thus, we have expanded the information on ORF2 at both sequence and structure levels. These findings may help to better understand the evolution and life cycle of HEV, but also facilitate the development of genetically engineered vaccines or antibodies.