No abstract

Hepatitis E virus (HEV) can account for up to a 30% mortality rate in pregnant women, with highest incidences reported for genotype 1 (gt1) HEV. Reasons contributing to adverse maternal-fetal outcome during pregnancy in HEV-infected pregnant women remain elusive in part due to the lack of a robust tissue culture model for some strains. Open reading frame (ORF4) was discovered overlapping ORF1 in gt1 HEV whose protein expression is regulated via an IRES-like RNA element. To experimentally determine whether gt3 HEV contains an ORF4-like gt1, gt1 and gt3 sequence comparisons were performed between the gt1 and the homologous gt3 sequence. To assess whether ORF4 protein could enhance gt3 replication, Huh7 cell lines constitutively expressing ORF4 were created and used to assess the replication of the Kernow-C1 gt3 and sar55 gt1 HEV. Virus stocks from transfected Huh7 cells with or without ORF4 were harvested and infectivity assessed via infection of HepG2/C3A cells. We also studied the replication of gt1 HEV in the ORF4-expressing tunicamycin-treated cell line. To directly show that HEV transcripts have productively replicated in the target cells, we assessed events at the single-cell level using indirect immunofluorescence and flow cytometry. Despite not naturally encoding ORF4, replication of gt3 HEV was enhanced by the presence of gt1 ORF4 protein. These results suggest that the function of ORF4 protein from gt1 HEV is transferrable, enhancing the replication of gt3 HEV. ORF4 may be utilized to enhance replication of difficult to propagate HEV genotypes in cell culture. IMPORTANCE: HEV is a leading cause of acute viral hepatitis (AVH) around the world. The virus is a threat to pregnant women, particularly during the second and third trimester of pregnancy. The factors enhancing virulence to pregnant populations are understudied. Additionally, field strains of HEV remain difficult to culture in vitro. ORF4 was recently discovered in gt1 HEV and is purported to play a role in pregnancy related pathology and enhanced replication. We present evidence that ORF4 protein provided in trans enhances the viral replication of gt3 HEV even though it does not encode ORF4 naturally in its genome. These data will aid in the development of cell lines capable of supporting replication of non-cell culture adapted HEV field strains, allowing viral titers sufficient for studying these strains in vitro. Furthermore, development of gt1/gt3 ORF4 chimeric virus may shed light on the role that ORF4 plays during pregnancy.

Hepatitis E virus is an important emerging pathogen producing a lethal impact on the pregnant population and immunocompromised patients. Starting in 1983, it has been described as the cause for acute hepatitis transmitted via the fecal–oral route. However, zoonotic and blood transfusion transmission of HEV have been reported in the past few decades, leading to the detailed research of HEV pathogenesis. The reason behind HEV being highly virulent to the pregnant population particularly during the third trimester, leading to maternal and fetal death, remains unknown. Various host factors (immunological, nutritional, hormonal) and viral factors have been studied to define the key determinants assisting HEV to be virulent in pregnant and immunocompromised patients. Similarly, chronic hepatitis is seen particularly in solid organ transplant patients, resulting in fatal conditions. This review describes recent advances in the immunopathophysiology of HEV infections in general, pregnant, and immunocompromised populations, and further elucidates the in vitro and in vivo models utilized to understand HEV pathogenesis.

Background Unlike the injectable vaccines, intranasal lipid nanoparticle (NP)-based adjuvanted vaccine is promising to protect against local infection and viral transmission. Infection of ferrets with SARS-CoV-2 results in typical respiratory disease and pathology akin to in humans, suggesting that the ferret model may be ideal for intranasal vaccine studies. Results We developed SARS-CoV-2 subunit vaccine containing both Spike receptor binding domain (S-RBD) and Nucleocapsid (N) proteins (NP-COVID-Proteins) or their mRNA (NP-COVID-mRNA) and NP-monosodium urate adjuvant. Both the candidate vaccines in intranasal vaccinated aged ferrets substantially reduced the replicating virus in the entire respiratory tract. Specifically, the NP-COVID-Proteins vaccine did relatively better in clearing the virus from the nasal passage early post challenge infection. The immune gene expression in NP-COVID-Proteins vaccinates indicated increased levels of mRNA of IFNα, MCP1 and IL-4 in lungs and nasal turbinates, and IFNγ and IL-2 in lungs; while proinflammatory mediators IL-1β and IL-8 mRNA levels in lungs were downregulated. In NP-COVID-Proteins vaccinated ferrets S-RBD and N protein specific IgG antibodies in the serum were substantially increased at both day post challenge (DPC) 7 and DPC 14, while the virus neutralizing antibody titers were relatively better induced by mRNA versus the proteins-based vaccine. In conclusion, intranasal NP-COVID-Proteins vaccine induced balanced Th1 and Th2 immune responses in the respiratory tract, while NP-COVID-mRNA vaccine primarily elicited antibody responses. Conclusions Intranasal NP-COVID-Proteins vaccine may be an ideal candidate to elicit increased breadth of immunity against SARS-CoV-2 variants.

Avian species often serve as transmission vectors and sources of recombination for viral infections due to their ability to travel vast distances and their gregarious behaviors. Recently a novel deltacoronavirus (DCoV) was identified in sparrows. Sparrow deltacoronavirus (SpDCoV), coupled with close contact between sparrows and swine carrying porcine deltacoronavirus (PDCoV) may facilitate recombination of DCoVs resulting in novel CoV variants. We hypothesized that the spike (S) protein or receptor-binding domain (RBD) from sparrow coronaviruses (SpCoVs) may enhance infection in poultry. We used recombinant chimeric viruses, which express S protein or the RBD of SpCoV (icPDCoV-SHKU17, and icPDCoV-RBDISU) on the genomic backbone of an infectious clone of PDCoV (icPDCoV). Chimeric viruses were utilized to infect chicken derived DF-1 cells, turkey poults, and embryonated chicken eggs (ECEs) to examine permissiveness, viral replication kinetics, pathogenesis and pathology. We demonstrated that DF-1 cells in addition to the positive control LLC-PK1 cells are susceptible to SpCoV spike- and RBD- recombinant chimeric virus infections. However, the replication of chimeric viruses in DF-1 cells, but not LLC-PK1 cells, was inefficient. Inoculated 8-day-old turkey poults appeared resistant to icPDCoV-, icPDCoV-SHKU17- and icPDCoV-RBDISU virus infections. In 5-day-old ECEs, significant mortality was observed in PDCoV inoculated eggs with less in the spike chimeras, while in 11-day-old ECEs there was no evidence of viral replication, suggesting that PDCoV is better adapted to cross species infection and differentiated ECE cells are not susceptible to PDCoV infection. Collectively, we demonstrate that the SpCoV chimeric viruses are not more infectious in turkeys, nor ECEs than wild type PDCoV. Therefore, understanding the cell and host factors that contribute to resistance to PDCoV and avian-swine chimeric virus infections may aid in the design of novel antiviral therapies against DCoVs.