Hepatitis E virus seropositivity in an ethnically diverse community blood donor population.

BACKGROUND AND OBJECTIVES: Hepatitis E virus (HEV) is an underrecognized and emerging infectious disease that may threaten the safety of donor blood supply in many parts of the world. We sought to elucidate whether our local community blood supply is at increased susceptibility for transmission of transfusion-associated HEV infections. MATERIALS AND METHODS: We screened 10,002 randomly selected donations over an 8-month period between 2017 and 2018 at the Stanford Blood Center for markers of HEV infection using commercial IgM/IgG serological tests and reverse transcriptase quantitative polymerase chain reaction assays (RT-qPCR). Donor demographic information, including gender, age, self-identified ethnicity, location of residence and recent travel, were obtained from the donor database and used to generate multivariate binary logistic regressions for risk factors of IgG seropositivity. RESULTS: A total of 10,002 blood donations from 7507 unique donors were screened, and there was no detectable HEV RNA by RT-qPCR. The overall seropositivity rate was 12.1% for IgG and 0.56% for IgM. Multivariate analysis of unique donors revealed a significantly higher risk of IgG seropositivity with increasing age, White/Asian ethnicities and residence in certain local counties. CONCLUSION: Although HEV IgG seroprevalence in the San Francisco Bay Area is consistent with ongoing infection, the screening of a large donor population did not identify any viraemic blood donors. While HEV is an underrecognized and emerging infection in other regions, there is no evidence to support routine blood screening for HEV in our local blood supply currently; however, periodic monitoring may still be required to assess the ongoing risk.

Fetal Loss in Pregnant Rabbits Infected with Genotype 3 Hepatitis E Virus Is Associated with Altered Inflammatory Responses, Enhanced Virus Replication, and Extrahepatic Virus Dissemination with Positive Correlations with Increased Estradiol Level.

Hepatitis E virus (HEV) causes adverse clinical outcomes in pregnant women, but the underlying mechanisms remain poorly understood. To delineate the mechanisms of pregnancy-associated adverse effects during HEV infection, we utilized a genotype 3 HEV from rabbit (HEV-3ra) and its cognate host (rabbits) to systematically investigate the clinical consequences, viral replication dynamics, and host immune and hormonal responses of HEV infection during pregnancy. We found a significant fetal loss of 23% in HEV-infected pregnant rabbits, indicating an early-stage miscarriage. HEV infection in pregnant rabbits was characterized by higher viral loads in feces, intestinal contents, liver, and spleen tissues, as well as a longer and earlier onset of viremia than in infected nonpregnant rabbits. HEV infection altered the pattern of cytokine gene expressions in the liver of pregnant rabbits and caused a transient increase of serum interferon gamma (IFN-γ) shortly after a notable increase in viral replication, which may contribute to early fetal loss. Histological lesions in the spleen were more pronounced in infected pregnant rabbits, although moderate liver lesions were seen in both infected pregnant and nonpregnant rabbits. Total bilirubin was elevated in infected pregnant rabbits. The serum levels of estradiol (E2) in HEV-infected pregnant rabbits were significantly higher than those in mock-infected pregnant rabbits at 14 days postinoculation (dpi) and correlated positively with higher viral loads in feces, liver, and spleen tissues at 28 dpi, suggesting that it may play a role in extrahepatic virus dissemination. The results have important implications for understanding the severe diseases associated with HEV infection during pregnancy. IMPORTANCE HEV causes adverse pregnancy outcomes, with a mortality rate of >30% in pregnant women, but the underlying mechanisms are poorly understood. In this study, we utilized HEV-3ra and its cognate host (pregnant rabbit) to delineate the potential underlying mechanisms of pregnancy-associated adverse outcomes during HEV infection. We found that infected pregnant rabbits had a fetal loss of 23%, which coincided with enhanced viral replication and an elevated systemic IFN-γ response, followed by longer viremia duration and extrahepatic viral dissemination. Estradiol levels were increased in infected pregnant rabbits and correlated positively with higher fecal viral shedding and higher viral loads in liver and spleen tissues. Infected pregnant rabbits had more pronounced splenic lesions, higher serum total bilirubin, and an altered cytokine gene expression profile in the liver. The results will contribute to our understanding of the mechanisms of HEV-associated adverse pregnancy outcomes.

A hepatitis B virus core antigen-based virus-like particle vaccine expressing SARS-CoV-2 B and T cell epitopes induces epitope-specific humoral and cell-mediated immune responses but confers limited protection against SARS-CoV-2 infection.

The hepatitis B virus core antigen (HBcAg) tolerates insertion of foreign epitopes and maintains its ability to self-assemble into virus-like particles (VLPs). We constructed a ∆HBcAg-based VLP vaccine expressing three predicted severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) B and T cell epitopes and determined its immunogenicity and protective efficacy. The recombinant ∆HBcAg-SARS-CoV-2 protein was expressed in Escherichia coli, purified, and shown to form VLPs. K18-hACE2 transgenic C57BL/6 mice were immunized intramuscularly with ∆HBcAg VLP control (n = 15) or ∆HBcAg-SARS-CoV-2 VLP vaccine (n = 15). One week after the 2nd booster and before virus challenge, five ∆HBcAg-SARS-CoV-2 vaccinated mice were euthanized to evaluate epitope-specific immune responses. There is a statistically significant increase in epitope-specific Immunoglobulin G (IgG) response, and statistically higher interleukin 6 (IL-6) and monocyte chemoattractant protein-1 (MCP-1) expression levels in ∆HBcAg-SARS-CoV-2 VLP-vaccinated mice compared to ∆HBcAg VLP controls. While not statistically significant, the ∆HBcAg-SARS-CoV-2 VLP mice had numerically more memory CD8+ T-cells, and 3/5 mice also had numerically higher levels of interferon gamma (IFN-γ) and tumor necrosis factor (TNF). After challenge with SARS-CoV-2, ∆HBcAg-SARS-CoV-2 immunized mice had numerically lower viral RNA loads in the lung, and slightly higher survival, but the differences are not statistically significant. These results indicate that the ∆HBcAg-SARS-CoV-2 VLP vaccine elicits epitope-specific humoral and cell-mediated immune responses but they were insufficient against SARS-CoV-2 infection.

Modulation of SOCS3 Levels via STAT3 and Estrogen-ERαp66 Signaling during Hepatitis E Virus Replication in Hepatocellular Carcinoma Cells.

Hepatitis E virus (HEV) infection usually results in a self-limiting acute disease; however, in infected pregnant women, it is associated with increased mortality and fulminant hepatic failure. Estrogen is known to be elevated during pregnancy, and estrogen signaling via classical estrogen receptor-ERα is known to regulate hepatocyte function and host innate immune response, including the STAT3 pathway. In this study, we investigated whether the estrogen classical signaling pathway via ERαp66 has any effect on STAT3 activation during HEV replication and HEV-induced IFN response. We first demonstrated that Huh7-S10-3 liver cells expressed the nonfunctional estrogen receptor ERαp36 isoform and lack the functional ERαp66 isoform. We further showed persistent phosphorylated-STAT3 levels in genotype 3 human HEV (Kernow P6 strain) RNA-transfected cells at later time points. In Huh7-S10-3 cells, estrogen at first-to-third trimester concentration (7.3 to 73 nM) did not significantly affect HEV replication; however, blocking of STAT3 activation led to a decrease in the HEV ORF2 protein level. Our mechanistic study revealed that STAT3 differentially regulates SOCS3 and type-III interferon (IFN) levels during HEV replication and the presence of estrogen-ERαp66 signaling stabilizes SOCS3 levels in vitro. We also demonstrate that HEV infection in pregnant and nonpregnant rabbits led to a significant increase in IFN response as measured by increased levels of IFN-stimulated-gene-15 (ISG15) mRNA levels irrespective of pregnancy status. Collectively, the results indicate that estrogen signaling and STAT3 regulate SOCS3 and IFN responses in vitro during HEV replication. The results have important implications for understanding HEV replication and HEV-induced innate immune response in pregnant women. IMPORTANCE Hepatitis E is usually a self-resolving acute disease; however, in pregnant women, HEV infection is associated with high mortality and fulminant hepatic failure. During pregnancy, estrogen levels are elevated, and in the liver, the estrogen receptor ERα is predominant and estrogen signaling is known to regulate hepatocyte metabolism and leptin-induced STAT3 levels. Viruses can module host innate immune response via STAT3. Therefore, in this study, we investigated whether STAT3 and estrogen-classical signaling via the ERαp66 pathway modulate HEV replication and HEV-induced innate immune response. We demonstrated that estrogen signaling did not affect HEV replication in human liver cells, but blocking of STAT3 activation reduced HEV capsid protein levels in human liver cells. We also showed that inhibition of STAT3 activation reduced SOCS3 levels, while the presence of the estrogen-ERαp66 signaling pathway stabilized SOCS3 levels. The results from this study will aid our understanding of the mechanism of HEV pathogenesis and immune response during pregnancy.

Two mutations in the ORF1 of genotype 1 hepatitis E virus enhance virus replication and may associate with fulminant hepatic failure.

Hepatitis E virus (HEV) infection in pregnant women has a high incidence of developing fulminant hepatic failure (FHF) with significant mortality. Multiple amino acid changes in genotype 1 HEV (HEV-1) are reportedly linked to FHF clinical cases, but experimental confirmation of the roles of these changes in FHF is lacking. By utilizing the HEV-1 indicator replicon and infectious clone, we generated 11 HEV-1 single mutants, each with an individual mutation, and investigated the effect of these mutations on HEV replication and infection in human liver cells. We demonstrated that most of the mutations actually impaired HEV-1 replication efficiency compared with the wild type (WT), likely due to altered physicochemical properties and structural conformations. However, two mutations, A317T and V1120I, significantly increased HEV-1 replication. Notably, these two mutations simultaneously occurred in 100% of 21 HEV-1 variants from patients with FHF in Bangladesh. We further created an HEV-1 A317T/V1120I double mutant and found that it greatly enhanced HEV replication, which may explain the rapid viral replication and severe disease. Furthermore, we tested the effect of these FHF-associated mutations on genotype 3 HEV (HEV-3) replication and found that all the mutants had a reduced level of replication ability and infectivity, which is not unexpected due to distinct infection patterns between HEV-1 and HEV-3. Additionally, we demonstrated that these FHF-associated mutations do not appear to alter their sensitivity to ribavirin (RBV), suggesting that ribavirin remains a viable option for antiviral therapy for patients with FHF. The results have important implications for understanding the mechanism of HEV-1-associated FHF.

Hepatitis E virus infects brain microvascular endothelial cells, crosses the blood-brain barrier, and invades the central nervous system.

Hepatitis E virus (HEV) is an important but understudied zoonotic virus causing both acute and chronic viral hepatitis. A proportion of HEV-infected individuals also developed neurological diseases such as Guillain-Barré syndrome, neuralgic amyotrophy, encephalitis, and myelitis, although the mechanism remains unknown. In this study, by using an in vitro blood-brain barrier (BBB) model, we first investigated whether HEV can cross the BBB and whether the quasi-enveloped HEV virions are more permissible to the BBB than the nonenveloped virions. We found that both quasi-enveloped and nonenveloped HEVs can similarly cross the BBB and that addition of proinflammatory cytokine tumor necrosis factor alpha (TNF-α) has no significant effect on the ability of HEV to cross the BBB in vitro. To explore the possible mechanism of HEV entry across the BBB, we tested the susceptibility of human brain microvascular endothelial cells lining the BBB to HEV infection and showed that brain microvascular endothelial cells support productive HEV infection. To further confirm the in vitro observation, we conducted an experimental HEV infection study in pigs and showed that both quasi-enveloped and nonenveloped HEVs invade the central nervous system (CNS) in pigs, as HEV RNA was detected in the brain and spinal cord of infected pigs. The HEV-infected pigs with detectable viral RNA in CNS tissues had histological lesions in brain and spinal cord and significantly higher levels of proinflammatory cytokines TNF-α and interleukin 18 than the HEV-infected pigs without detectable viral RNA in CNS tissues. The findings suggest a potential mechanism of HEV-associated neuroinvasion.

Progesterone-Mediated Enhancement of Hepatitis E Virus Replication in Human Liver Cells.

Progesterone is crucial for the maintenance of pregnancy. During pregnancy hepatitis E virus (HEV) infection is associated with increased fulminant hepatic failure and mortality rates. In this study, we determined whether progesterone modulates HEV replication and HEV-induced innate cytokine response in Huh7-S10-3 human liver cells. We first demonstrated that Huh7-S10-3 liver cells expressed SH3-domain-containing progesterone receptor membrane component (PGRMC)1/2 receptors involved in the progesterone nonclassical signaling pathway, while the classical progesterone receptor isoforms progesterone receptor-A and -B protein levels were undetectable. We showed that the genotype 3 HEV (strain P6) induced mRNA expression of type III interferon (IFN-λ1), but not other innate cytokines in Huh7-S10-3 cells. Pretreatment with progesterone at concentrations of 80 nM, 160 nM, or 480 nM, which are the physiological concentrations typically seen in the first- to third-trimester during pregnancy, significantly increased HEV replication in Huh7-S10-3 cells. However, pretreatment of cells with progesterone (80 nM) did not affect the level of HEV-induced IFN-λ1 mRNA expression. We further showed that loss of PGRMC1/2 receptors by small interfering RNA (siRNA) knockdown leads to an increase in HEV-induced IFN-λ1 expression levels at early time points via the extracellular signal-regulated kinase pathway and thus resulted in a reduced level of HEV replication. Collectively, the results indicated that progesterone-mediated modulation of HEV replication in human liver cells is plausibly through SH3-domain containing proteins such as PGRMC1/2, but not likely through immunomodulation of HEV-induced interferon response in liver cells. The results have important implications in understanding the underlying mechanisms of high mortality and fulminant hepatitis in HEV-infected pregnant women. IMPORTANCE Hepatitis E is usually a self-limiting acute disease; however, during pregnancy, a severe form of fulminant hepatic failure and high mortality rate are associated with hepatitis E virus (HEV) infection. Increased levels of progesterone and HEV RNA are observed in pregnant women with fulminant hepatic failures. Since progesterone is crucial for maintenance of pregnancy, we investigated the potential role of progesterone in HEV replication and disease pathogenesis. We demonstrated that progesterone at a concentration seen during pregnancy enhances HEV replication in human liver cells, but did not modulate HEV-induced interferon response in human liver cells. We also showed that loss of the progesterone nonclassical receptor, progesterone receptor membrane component (PGRMC)1/2, leads to a reduced level of HEV replication and an increased level of HEV-induced type III interferon (IFN-λ1) mRNA expression via the extracellular signal-regulated kinase pathway. The results from this study will aid our understanding of the underlying mechanism of pathogenesis and HEV-associated severe disease during pregnancy.

Killed whole-genome reduced-bacteria surface-expressed coronavirus fusion peptide vaccines protect against disease in a porcine model.

As the coronavirus disease 2019 (COVID-19) pandemic rages on, it is important to explore new evolution-resistant vaccine antigens and new vaccine platforms that can produce readily scalable, inexpensive vaccines with easier storage and transport. We report here a synthetic biology-based vaccine platform that employs an expression vector with an inducible gram-negative autotransporter to express vaccine antigens on the surface of genome-reduced bacteria to enhance interaction of vaccine antigen with the immune system. As a proof-of-principle, we utilized genome-reduced Escherichia coli to express SARS-CoV-2 and porcine epidemic diarrhea virus (PEDV) fusion peptide (FP) on the cell surface, and evaluated their use as killed whole-cell vaccines. The FP sequence is highly conserved across coronaviruses; the six FP core amino acid residues, along with the four adjacent residues upstream and the three residues downstream from the core, are identical between SARS-CoV-2 and PEDV. We tested the efficacy of PEDV FP and SARS-CoV-2 FP vaccines in a PEDV challenge pig model. We demonstrated that both vaccines induced potent anamnestic responses upon virus challenge, potentiated interferon-γ responses, reduced viral RNA loads in jejunum tissue, and provided significant protection against clinical disease. However, neither vaccines elicited sterilizing immunity. Since SARS-CoV-2 FP and PEDV FP vaccines provided similar clinical protection, the coronavirus FP could be a target for a broadly protective vaccine using any platform. Importantly, the genome-reduced bacterial surface-expressed vaccine platform, when using a vaccine-appropriate bacterial vector, has potential utility as an inexpensive, readily manufactured, and rapid vaccine platform for other pathogens.

Construction and efficacy evaluation of novel swine leukocyte antigen (SLA) class I and class II allele-specific poly-T cell epitope vaccines against porcine reproductive and respiratory syndrome virus.

Porcine reproductive and respiratory syndrome virus (PRRSV) causes an economically important global swine disease. Here we report the development of subunit PRRSV-2 vaccines by expressing swine leucocyte antigen (SLA) class I and class II allele-specific epitope antigens in a robust adenovirus vector. SLA I-specific CD8 and SLA II-specific CD4 T cell epitopes of PRRSV-2 NADC20 were predicted in silico. Stable murine leukaemia cell lines (RMA-S), which are TAP-deficient and lacking endogenous class I epitope loading, were established to express different SLA I alleles. The binding stability of PRRSV T cell epitope peptides with SLA I alleles expressed on RMA-S cells was characterized. Two PRRSV poly-T cell epitope peptides were designed. NADC20-PP1 included 39 class I epitopes, consisting of 8 top-ranked epitopes specific to each of 5 SLA I alleles, and fused to 5 class II epitopes specific to SLA II alleles. NADC20-PP2, a subset of PP1, included two top-ranked class I epitopes specific to each of the five SLA I alleles. Two vaccine candidates, Ad-NADC20-PP1 and Ad-NADC20-PP2, were constructed by expressing the polytope peptides in a replication-incompetent human adenovirus 5 vector. A vaccination and challenge study in 30 piglets showed that animals vaccinated with the vaccines had numerically lower gross and histopathology lung lesions, and numerically lower PRRSV RNA loads in lung and serum after challenge compared to the controls, although there was no statistical significance. The results suggested that the Ad-NADC20-PP1 and Ad-NADC20-PP2 vaccines provided little or no protection, further highlighting the tremendous challenges faced in developing an effective subunit PRRSV-2 vaccine.

Virus-like particle vaccine with B-cell epitope from porcine epidemic diarrhea virus (PEDV) incorporated into hepatitis B virus core capsid provides clinical alleviation against PEDV in neonatal piglets through lactogenic immunity.

Porcine epidemic diarrhea virus (PEDV) has had a negative economic impact on the global swine industry for decades since its first emergence in the 1970s in Europe. In 2013, PEDV emerged for the first time in the United States, causing immense economic losses to the swine industry. Efforts to protect U.S. swine herds from PEDV infection and limit PEDV transmission through vaccination had only limited success so far. Following the previous success in our virus-like particle (VLP) based vaccine in mouse model, in this study we determined the immunogenicity and protective efficacy of a VLP-based vaccine containing B-cell epitope 748YSNIGVCK755 from the spike protein of PEDV incorporated into the hepatitis B virus core capsid (HBcAg), in a comprehensive pregnant gilt vaccination and piglet challenge model. The results showed that the vaccine was able to induce significantly higher virus neutralization response in gilt milk, and provide alleviation of clinical signs for piglets experimentally infected with PEDV. Piglets from pregnant gilt that was vaccinated with the VLP vaccine had faster recovery from the clinical disease, less small intestinal lesions, and higher survival rate at 10 days post-challenge (DPC).

Swine hepatitis E virus: Cross-species infection, pork safety and chronic infection.

Swine hepatitis E virus (swine HEV) belongs to the species Orthohepevirus A within the genus Orthohepevirus in the family Hepeviridae. Four different genotypes of swine HEV within the species Orthohepevirus A have been identified so far from domesticated and wild swine population: genotypes 3 (HEV-3) and 4 (HEV-4) swine HEVs are zoonotic and infect humans, whereas HEV-5 and HEV-6 are only identified from swine. As a zoonotic agent, swine HEV is an emerging public health concern in many industrialized countries. Pigs are natural reservoir for HEV, consumption of raw or undercooked pork is an important route of foodborne HEV transmission. Occupational risks such as direct contact with infected pigs also increase the risk of HEV transmission in humans. Cross-species infection of HEV-3 and HEV-4 have been documented under experimental and natural conditions. Both swine HEV-3 and swine HEV-4 infect non-human primates, the surrogates of man. Swine HEV, predominantly HEV-3, can establish chronic infection in immunocompromised patients especially in solid organ transplant recipients. The zoonotic HEV-3, and to lesser extent HEV-4, have also been shown to cause neurological diseases and kidney injury. In this review, we focus on the epidemiology of swine HEV, host and viral determinants influencing cross-species HEV infection, zoonotic infection and its associated pork safety concern, as well as swine HEV-associated chronic infection and neurological diseases.

Hepatitis E Virus in Pigs from Slaughterhouses, United States, 2017-2019.

Hepatitis E virus (HEV) RNA was detected in 6.3% and HEV IgG in 40% of 5,033 serum samples from market-weight pigs at 25 slaughterhouses in 10 US states. The prevalent HEV genotype was zoonotic genotype 3, group 2. Blood of HEV-viremic pigs from slaughterhouses may contaminate pork supply chains.

The U-Rich Untranslated Region of the Hepatitis E Virus Induces Differential Type I and Type III Interferon Responses in a Host Cell-Dependent Manner.

Hepatitis E virus (HEV), a single-strand positive-sense RNA virus, is an understudied but important human pathogen. The virus can establish infection at a number of host tissues, including the small intestine and liver, causing acute and chronic hepatitis E as well as certain neurological disorders. The retinoic acid-inducible gene I (RIG-I) pathway is essential to induce the interferon (IFN) response during HEV infection. However, the pathogen-associated motif patterns (PAMPs) in the HEV genome that are recognized by RIG-I remain unknown. In this study, we first identified that HEV RNA PAMPs derived from the 3' untranslated region (UTR) of the HEV genome induced higher levels of IFN mRNA, interferon regulatory factor-3 (IRF3) phosphorylation, and nuclear translocation than the 5' UTR of HEV. We revealed that the U-rich region in the 3' UTR of the HEV genome acts as a potent RIG-I PAMP, while the presence of poly(A) tail in the 3' UTR further increases the potency. We further demonstrated that HEV UTR PAMPs induce differential type I and type III IFN responses in a cell type-dependent fashion. Predominant type III IFN response was observed in the liver tissues of pigs experimentally infected with HEV as well as in HEV RNA PAMP-induced human hepatocytes in vitro In contrast, HEV RNA PAMPs induced a predominant type I IFN response in swine enterocytes. Taken together, the results from this study indicated that the IFN response during HEV infection depends both on viral RNA motifs and host target cell types. The results have important implications in understanding the mechanism of HEV pathogenesis.IMPORTANCE Hepatitis E virus (HEV) is an important human pathogen causing both acute and chronic viral hepatitis E infection. Currently, the mechanisms of HEV replication and pathogenesis remain poorly understood. The innate immune response acts as the first line of defense during viral infection. The retinoic acid-inducible gene I (RIG-I)-mediated interferon (IFN) response has been implicated in establishing antiviral response during HEV infection, although the HEV RNA motifs that are recognized by RIG-I are unknown. This study identified that the U-rich region in the 3' untranslated region (UTR) of the HEV genome acts as a potent RIG-I agonist compared to the HEV 5' UTR. We further revealed that the HEV RNA pathogen-associated motif patterns (PAMPs) induced a differential IFN response in a cell type-dependent manner: a predominantly type III IFN response in hepatocytes, and a predominantly type I IFN response in enterocytes. These data demonstrate the complexity by which both host and viral factors influence the IFN response during HEV infection.

Hepatitis E virus: reasons for emergence in humans.

Hepatitis E virus (HEV) infects both humans and other animal species. Recently, we have seen a steady increase in autochthonous cases of human HEV infection in certain areas especially in Europe, and large outbreaks in several African countries among the displaced population. This mini-review critically analyzes potential host, environmental, and viral factors that may be associated with the emergence of hepatitis E in humans. The existence of numerous HEV reservoir animals such as pig, deer and rabbit results in human exposure to infected animals via direct contact or through animal meat consumption. Contamination of drinking, irrigation and coastal water by animal and human wastes lead to emergence of endemic cases in industrialized countries and outbreaks in displaced communities especially in war-torn countries.

Evaluation of the pathogenicity of mammalian orthoreovirus type 3 (MRV3) in germ-free gnotobiotic pigs and of the efficacy of an inactivated vaccine against MRV3 infection in neonatal conventional piglets.

A novel U.S. strain of mammalian orthoreovirus type 3 (MRV3) isolated from diarrheic pigs in 2015 was reportedly highly pathogenic in pigs. In this study, we first developed an inactivated MRV3 vaccine and determined its protective efficacy against MRV3 infection in conventional neonatal piglets. A pathogenicity study was also conducted in gnotobiotic pigs to further assess the pathogenicity of MRV3. To evaluate if piglets could be protected against MRV3 infection after immunization of pregnant sows with an inactivated MRV3 vaccine, pregnant sows were vaccinated with 2 or 3 doses of the vaccine or with PBS buffer. Four-day-old piglets born to vaccinated and unvaccinated sows were subsequently challenged with MRV3. The results showed that piglets born from vaccinated sows had lower levels of fecal viral RNA shedding at 1, 3, and 4 days post-challenge, suggesting that the inactivated MRV3 vaccine can reduce MRV3 replication. Surprisingly, although the conventional piglets were infected, they did not develop severe enteric disease as reported previously. Therefore, in an effort to further definitively assess the pathogenicity of MRV3, we experimentally infected gnotobiotic pigs, a more sensitive model for pathogenicity study, with the wild-type MRV3 virus. The infected gnotobiotic piglets all survived and exhibited only very mild diarrhea in some pigs. Taken together, the results indicate that the novel strain of MRV3 recently isolated in the United States infected but caused only very mild diarrhea in pigs, and that maternal immunity acquired from sows vaccinated with an inactivated vaccine can reduce MRV3 replication in neonatal pigs.

Infection Dynamics of Hepatitis E Virus in Wild-Type and Immunoglobulin Heavy Chain Knockout JH-/- Gnotobiotic Piglets.

Hepatitis E virus (HEV), the causative agent of hepatitis E, is an important but incompletely understood pathogen causing high mortality during pregnancy and leading to chronic hepatitis in immunocompromised individuals. The underlying mechanisms leading to hepatic damage remain unknown; however, the humoral immune response is implicated. In this study, immunoglobulin (Ig) heavy chain JH-/- knockout gnotobiotic pigs were generated using CRISPR/Cas9 technology to deplete the B-lymphocyte population, resulting in an inability to generate a humoral immune response to genotype 3 HEV infection. Compared to wild-type gnotobiotic piglets, the frequencies of B lymphocytes in the Ig heavy chain JH-/- knockouts were significantly lower, despite similar levels of other innate and adaptive T-lymphocyte cell populations. The dynamic of acute HEV infection was subsequently determined in heavy chain JH-/- knockout and wild-type gnotobiotic pigs. The data showed that wild-type piglets had higher viral RNA loads in feces and sera compared to the JH-/- knockout pigs, suggesting that the Ig heavy chain JH-/- knockout in pigs actually decreased the level of HEV replication. Both HEV-infected wild-type and JH-/- knockout gnotobiotic piglets developed more pronounced lymphoplasmacytic hepatitis and hepatocellular necrosis lesions than other studies with conventional pigs. The HEV-infected JH-/- knockout pigs also had significantly enlarged livers both grossly and as a ratio of liver/body weight compared to phosphate-buffered saline-inoculated groups. This novel gnotobiotic pig model will aid in future studies into HEV pathogenicity, an aspect which has thus far been difficult to reproduce in the available animal model systems.IMPORTANCE According to the World Health Organization, approximately 20 million HEV infections occur annually, resulting in 3.3 million cases of hepatitis E and >44,000 deaths. The lack of an efficient animal model that can mimic the full-spectrum of infection outcomes hinders our ability to delineate the mechanism of HEV pathogenesis. Here, we successfully generated immunoglobulin heavy chain JH-/- knockout gnotobiotic pigs using CRISPR/Cas9 technology, established a novel JH-/- knockout and wild-type gnotobiotic pig model for HEV, and systematically determined the dynamic of acute HEV infection in gnotobiotic pigs. It was demonstrated that knockout of the Ig heavy chain in pigs decreased the level of HEV replication. Infected wild-type and JH-/- knockout gnotobiotic piglets developed more pronounced HEV-specific lesions than other studies using conventional pigs, and the infected JH-/- knockout pigs had significantly enlarged livers. The availability of this novel model will facilitate future studies of HEV pathogenicity.

Vaccination of sows with a dendritic cell-targeted porcine epidemic diarrhea virus S1 protein-based candidate vaccine reduced viral shedding but exacerbated gross pathological lesions in suckling neonatal piglets.

Porcine epidemic diarrhea virus (PEDV) poses a serious threat to swine worldwide as evidenced by its recent introduction into the USA and the devastating economic impact it caused to the USA swine industry. Commercial vaccines against PEDV are available but their efficacies are inadequate. Therefore, vaccines with improved efficacy are needed to effectively control PEDV infections. We previously determined the immunogenicity of a novel dendritic cell (DC)-targeted PEDV S1 protein-based subunit vaccine in weaned piglets in which the PEDV antigen was targeted to DCs through a porcine Langerin-specific antibody. In this study, we evaluated the protective efficacy of this DC-targeting vaccine by immunizing sows at 5 and 2 weeks prior to farrowing and by challenging the 5-day-old piglets with PEDV. The results showed that immunization of sow with DC-targeted PEDV vaccine did not eliminate faecal virus shedding in piglets but significantly reduced faecal viral RNA levels in the early days after virus challenge. The vaccine also reduced the amount of PEDV antigen in intestinal tissues presented with intestinal villi regrowth. However, the DC-targeted vaccine neither mitigated PEDV clinical signs nor affected viral RNA loads in intestinal tissues of piglets. In the vaccinated sow, DC-targeted PEDV vaccine enhanced T helper 1-like cluster of differentiation (CD)4 T cell responses and induced IgG but not IgA-specific immune responses. The suckling piglets in the DC-targeted vaccine group showed increased gross pathological lesions in the small intestine. Results in this study provide insights into the effects of sow cellular immune responses to PEDV infection in suckling piglets.

Protective efficacy of a virus-vectored multi-component vaccine against porcine reproductive and respiratory syndrome virus, porcine circovirus type 2 and swine influenza virus.

Porcine reproductive and respiratory syndrome virus (PRRSV), porcine circovirus type 2 (PCV2) and swine influenza virus (SIV) are three of the most economically important swine pathogens, causing immense economic losses to the global swine industry. Monovalent commercial vaccines against each of the three viruses are routinely used in pig farms worldwide. A trivalent vaccine against all three pathogens would greatly simplify the vaccination programme and reduce the financial burden to the swine industry. In this study, by using an attenuated strain of PRRSV (strain DS722) as a live virus vector, we generated a multi-component vaccine virus, DS722-SIV-PCV2, which expresses the protective antigens from SIV and PCV2. The DS722-SIV-PCV2 trivalent vaccine virus replicates well, and expresses PCV2 capsid and SIV HA proteins in vitro. A subsequent vaccination and challenge study in 48 pigs revealed that the DS722-SIV-PCV2-vaccinated pigs had significantly reduced lung lesions and viral RNA loads when challenged with PRRSV. Upon challenge with PCV2, the vaccinated pigs had partially reduced lymphoid lesions and viral DNA loads, and when challenged with SIV the vaccinated pigs had significantly reduced acute respiratory sign scores. The results from this study demonstrate the potential of DS722-SIV-PCV2 as a candidate trivalent vaccine, and also shed light on exploring PRRSV as a potential live virus vaccine vector.

ISG15 Modulates Type I Interferon Signaling and the Antiviral Response during Hepatitis E Virus Replication.

Hepatitis E virus (HEV), a single-stranded positive-sense RNA virus, generally causes self-limiting acute viral hepatitis, although chronic HEV infection has recently become a significant clinical problem in immunocompromised individuals, especially in solid-organ transplant recipients. Innate immunity, via the type I interferon (IFN) response, plays an important role during the initial stages of a viral infection. IFN-stimulated gene 15 (ISG15), an IFN-induced ubiquitin-like protein, is known to have an immunomodulatory role and can have a direct antiviral effect on a wide spectrum of virus families. In the present study, we investigated the antiviral effect as well as the potential immunomodulatory role of ISG15 during HEV replication. The results revealed that HEV induced high levels of ISG15 production both in vitro (Huh7-S10-3 liver cells) and in vivo (liver tissues from HEV-infected pigs); however, ISG15 is not required for virus replication. We also demonstrated that ISG15 silencing potentiates enhanced type I IFN-mediated signaling, resulting in an increase in the type I IFN-mediated antiviral effect during HEV replication. This observed enhanced type I IFN signaling correlated with an increase in IFN-stimulated gene expression levels during HEV replication. Furthermore, we showed that PKR and OAS1 played important roles in the ISG15-mediated type I IFN sensitivity of HEV. Taken together, the results from this study suggest that ISG15 plays an important immunomodulatory role and regulates HEV sensitivity to exogenous type I IFN.IMPORTANCE Hepatitis E virus (HEV) infection typically causes self-limiting acute viral hepatitis. However, chronic HEV infection has recently become a significant clinical problem in immunocompromised patients. Pegylated interferon (IFN) has been used to treat chronic HEV infection in solid-organ transplant patients with some success. However, the mechanism behind the type I IFN-mediated antiviral effect against HEV remains unclear. This report demonstrates that ISG15 induced by HEV replication in Huh7-S10-3 human liver cells plays an immunomodulatory role by negatively regulating type I IFN signaling and, thus, HEV sensitivity to type I IFN. Our results also show that PKR and OAS1 play important roles in the ISG15-mediated type I IFN sensitivity of HEV.