Infectious hepatitis E virus is associated with the mature sperm head.

Hepatitis E virus (HEV) is the leading cause of acute viral hepatitis worldwide. HEV associated pregnancy mortality has been reported as up to 30% in humans. Recent findings suggest HEV may elicit effects directly in the reproductive system with HEV protein found in the testis, viral RNA in semen, and viral replication occurring in placental cell types. Using a natural host model for HEV infection, pigs, we demonstrate infectious HEV within the mature spermatozoa and altered sperm viability from HEV infected pigs. HEV isolated from sperm remained infectious suggesting a potential transmission route via sexual partners. Our findings suggest that HEV should be explored as a possible sexually transmittable disease. Our findings propose that infection routes outside of oral and intravenous infection need to be considered for their potential to contribute to higher mortality in HEV infections when pregnancy is involved and in HEV disease in general.

Enhanced mucosal immune responses and reduced viral load in the respiratory tract of ferrets to intranasal lipid nanoparticle-based SARS-CoV-2 proteins and mRNA vaccines.

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-1ß 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.

Porcine Deltacoronavirus Infection and Transmission in Poultry, United States1.

Coronaviruses cause respiratory and gastrointestinal diseases in diverse host species. Deltacoronaviruses (DCoVs) have been identified in various songbird species and in leopard cats in China. In 2009, porcine deltacoronavirus (PDCoV) was detected in fecal samples from pigs in Asia, but its etiologic role was not identified until 2014, when it caused major diarrhea outbreaks in swine in the United States. Studies have shown that PDCoV uses a conserved region of the aminopeptidase N protein to infect cell lines derived from multiple species, including humans, pigs, and chickens. Because PDCoV is a potential zoonotic pathogen, investigations of its prevalence in humans and its contribution to human disease continue. We report experimental PDCoV infection and subsequent transmission among poultry. In PDCoV-inoculated chicks and turkey poults, we observed diarrhea, persistent viral RNA titers from cloacal and tracheal samples, PDCoV-specific serum IgY antibody responses, and antigen-positive cells from intestines.

Development of fatty liver disease model using high cholesterol and low choline diet in white leghorn chickens.

Nonalcoholic fatty liver disease (NAFLD), which shows similar symptoms as fatty liver hemorrhage syndrome (FLHS) in chickens, is the most common cause of chronic liver disease and cancer in humans. NAFLD patients and FLHS in chickens have demonstrated severe liver disorders when infected by emerging strains of human hepatitis E virus (HEV) and avian HEV, respectively. We sought to develop a fatty liver disease chicken model by altering the diet of 3-week-old white leghorn chickens. The high cholesterol, and low choline (HCLC) diet included 7.6% fat with additional 2% cholesterol and 800 mg/kg choline in comparison to 5.3% fat, and 1,300 mg/kg choline in the regular diet. Our diet induced fatty liver avian model successfully recapitulates the clinical features seen during NAFLD in humans and FLHS in chickens, including hyperlipidemia and hepatic steatosis, as indicated by significantly higher serum triglycerides, serum cholesterol, liver triglycerides, cholesterol, and fatty acids. By developing this chicken model, we expect to provide a platform to explore the role of lipids in the liver pathology linked with viral infections and contribute to the development of prophylactic interventions.

Rat hepatitis E virus cross-species infection and transmission in pigs.

Strains of Rocahepevirus ratti, an emerging hepatitis E virus (HEV), have recently been found to be infectious to humans. Rats are a primary reservoir of the virus; thus, it is referred to as "rat HEV". Rats are often found on swine farms in close contact with pigs. Our goal was to determine whether swine may serve as a transmission host for zoonotic rat HEV by characterizing an infectious cDNA clone of a zoonotic rat HEV, strain LCK-3110, in vitro and in vivo. RNA transcripts of LCK-3110 were constructed and assessed for their replicative capacity in cell culture and in gnotobiotic pigs. Fecal suspension from rat HEV-positive gnotobiotic pigs was inoculated into conventional pigs co-housed with naïve pigs. Our results demonstrated that capped RNA transcripts of LCK-3110 rat HEV replicated in vitro and successfully infected conventional pigs that transmit the virus to co-housed animals. The infectious clone of rat HEV may afford an opportunity to study the genetic mechanisms of rat HEV cross-species infection and tissue tropism.

SARS-CoV-2 Serological Investigation of White-Tailed Deer in Northeastern Ohio.

Coronaviruses are known to cross species barriers, and spill over among animals, from animals to humans, and vice versa. SARS-CoV-2 emerged in humans in late 2019. It is now known to infect numerous animal species, including companion animals and captive wildlife species. Experimental infections in other animals have established that many species are susceptible to infection, with new ones still being identified. We have developed an enzyme-linked immunosorbent assay (ELISA) for detecting antibodies to SARS-CoV-2 nucleocapsid (N) and spike (S) proteins, that is both sensitive and specific. It can detect S antibodies in sera at dilutions greater than 1:10,000, and does not cross-react with antibodies to the other coronaviruses tested. We used the S antibody ELISA to test serum samples collected from 472 deer from ten sites in northeastern Ohio between November 2020 and March 2021, when the SARS-CoV-2 pandemic was first peaking in humans in Ohio, USA. Antibodies to SARS-CoV-2 were found in serum samples from every site, with an overall positivity rate of 17.2%; we further compared the viral neutralizing antibody titers to our ELISA results. These findings demonstrate the need to establish surveillance programs to monitor deer and other susceptible wildlife species globally.

Characterization of the Cross-Species Transmission Potential for Porcine Deltacoronaviruses Expressing Sparrow Coronavirus Spike Protein in Commercial Poultry.

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.

Ectopic Expression of Genotype 1 Hepatitis E Virus ORF4 Increases Genotype 3 HEV Viral Replication in Cell Culture.

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.

Comparative Transcriptome Profiling of Human and Pig Intestinal Epithelial Cells after Porcine Deltacoronavirus Infection.

Porcine deltacoronavirus (PDCoV) is an emerging infectious disease of swine with zoonotic potential. Phylogenetic analysis suggests that PDCoV originated recently from a host-switching event between birds and mammals. Little is known about how PDCoV interacts with its differing hosts. Human-derived cell lines are susceptible to PDCoV infection. Herein, we compare the gene expression profiles of an established host swine cells to potential emerging host human cells after infection with PDCoV. Cell lines derived from intestinal lineages were used to reproduce the primary sites of viral infection in the host. Porcine intestinal epithelial cells (IPEC-J2) and human intestinal epithelial cells (HIEC) were infected with PDCoV. RNA-sequencing was performed on total RNA extracted from infected cells. Human cells exhibited a more pronounced response to PDCoV infection in comparison to porcine cells with more differentially expressed genes (DEGs) in human, 7486, in comparison to pig cells, 1134. On the transcriptional level, the adoptive host human cells exhibited more DEGs in response to PDCoV infection in comparison to the primary pig host cells, where different types of cytokines can control PDCoV replication and virus production. Key immune-associated DEGs and signaling pathways are shared between human and pig cells during PDCoV infection. These included genes related to the NF-kappa-B transcription factor family, the interferon (IFN) family, the protein-kinase family, and signaling pathways such as the apoptosis signaling pathway, JAK-STAT signaling pathway, inflammation/cytokine-cytokine receptor signaling pathway. MAP4K4 was unique in up-regulated DEGs in humans in the apoptosis signaling pathway. While similarities exist between human and pig cells in many pathways, our research suggests that the adaptation of PDCoV to the porcine host required the ability to down-regulate many response pathways including the interferon pathway. Our findings provide an important foundation that contributes to an understanding of the mechanisms of PDCoV infection across different hosts. To our knowledge, this is the first report of transcriptome analysis of human cells infected by PDCoV.

Notch-Nrf2 axis: regulation of Nrf2 gene expression and cytoprotection by notch signaling.

The Notch signaling pathway enables regulation and control of development, differentiation, and homeostasis through cell-cell communication. Our investigation shows that Notch signaling directly activates the Nrf2 stress adaptive response pathway through recruitment of the Notch intracellular domain (NICD) transcriptosome to a conserved Rbpjκ site in the promoter of Nrf2. Stimulation of Notch signaling through Notch ligand expression in cells and by overexpression of the NICD in Rosa(NICD/-)::AlbCre mice in vivo induces expression of Nrf2 and its target genes. Continuous and transient NICD expression in the liver produces a Notch-dependent cytoprotective response through direct transcriptional activation of Nrf2 signaling to rescue mice from acute acetaminophen toxicity. This response can be reversed upon genetic disruption of Nrf2. Morphological studies showed that the characteristic phenotype of high-density intrahepatic bile ducts and enlarged liver in Rosa(NICD/-)::AlbCre mice could be at least partially reversed after Nrf2 disruption. Furthermore, the liver and bile duct phenotypes could be recapitulated with constitutive activation of Nrf2 signaling in Keap1(F/F)::AlbCre mice. It appears that Notch-to-Nrf2 signaling is another important determinant in liver development and function and promotes cell-cell cytoprotective signaling responses.