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.

Ribavirin Treatment Failure-Associated Mutation, Y1320H, in the RNA-Dependent RNA Polymerase of Genotype 3 Hepatitis E Virus (HEV) Enhances Virus Replication in a Rabbit HEV Infection Model.

Chronic hepatitis E virus (HEV) infection has become a significant clinical problem that requires treatment in immunocompromised individuals. In the absence of an HEV-specific antiviral, ribavirin (RBV) has been used off-label, but treatment failure may occur due to mutations in the viral RNA-dependent RNA polymerase (RdRp), including Y1320H, K1383N, and G1634R. Chronic hepatitis E is mostly caused by zoonotic genotype 3 HEV (HEV-3), and HEV variants from rabbits (HEV-3ra) are closely related to human HEV-3. Here, we explored whether HEV-3ra, along with its cognate host, can serve as a model to study RBV treatment failure-associated mutations observed in human HEV-3-infected patients. By utilizing the HEV-3ra infectious clone and indicator replicon, we generated multiple single mutants (Y1320H, K1383N, K1634G, and K1634R) and a double mutant (Y1320H/K1383N) and assessed the role of mutations on replication and antiviral activity of HEV-3ra in cell culture. Furthermore, we also compared the replication of the Y1320H mutant with the wild-type HEV-3ra in experimentally infected rabbits. Our in vitro analyses revealed that the effects of these mutations on rabbit HEV-3ra are altogether highly consistent with those on human HEV-3. Importantly, we found that the Y1320H enhances virus replication during the acute stage of HEV-3ra infection in rabbits, which corroborated our in vitro results showing an enhanced viral replication of Y1320H. Taken together, our data suggest that HEV-3ra and its cognate host is a useful and relevant naturally occurring homologous animal model to study the clinical relevance of antiviral-resistant mutations observed in human HEV-3 chronically-infected patients. IMPORTANCE HEV-3 causes chronic hepatitis E that requires antiviral therapy in immunosuppressed individuals. RBV is the main therapeutic option for chronic hepatitis E as an off-label use. Several amino acid changes, including Y1320H, K1383N, and G1634R, in the RdRp of human HEV-3 have reportedly been associated with RBV treatment failure in chronic hepatitis E patients. In this study, we utilized an HEV-3ra from rabbit and its cognate host to investigate the effect of these RBV treatment failure-associated HEV-3 RdRp mutations on viral replication efficiency and antiviral susceptibility. The in vitro data using rabbit HEV-3ra was highly comparable to those from human HEV-3. We demonstrated that the Y1320H mutation significantly enhanced HEV-3ra replication in cell culture and enhanced virus replication during the acute stage of HEV-3ra infection in rabbits. The rabbit HEV-3ra infection model should be useful in delineating the role of human HEV-3 RBV treatment failure-associated mutations in antiviral resistance.

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.

PAPA-like syndrome with heterozygous mutation in the MEFV gene.

A patient presented with a history of recurrent pyoderma gangrenosum, arthritis and extensive acne, prompting a genetic workup for PAPA syndrome. An MEFV mutation was identified and a change in therapeutic strategy from anakinra to colchicine was successful. Click https://www.wileyhealthlearning.com/#/online-courses/b52447c0-1d37-472d-b0c0-7817352d6f68 for the corresponding questions to this CME article.

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.

Skin Cancer Excision Is More Efficient and Cost Effectivein a Specialist Secondary Care Service.

Aim To compare the relative efficiencies of skin excisions in primary and secondary care. Methods We compared the benign: malignant ratio for specimens referred by General Practice, General Surgery and the Skin Cancer Service to the regional pathology laboratory over one month. We used cost minimization analysis to compare the relative efficiencies of the services. Results 620 excisions were received: 139 from General Practice, 118 from General Surgery and 363 from the Skin Cancer Service. The number (%) of malignant lesions was 13 (9.4%) from General Practice, 18 (15.2%) from General Surgery and 137 (37.7%) from the Skin Cancer Service. Excision was cheaper in General Practice at €84.58 as compared to €97.49 in the hospital day surgical unit. However, the cost per malignant lesion excised was €1779.80 in general practice versus €381.78 in the Skin Cancer Service. Conclusion Our results indicate that moving skin cancer treatment to General Practice may result in an excess of benign excisions and therefore be both less efficient and less cost effective.

Dissecting the potential role of hepatitis E virus ORF1 nonstructural gene in cross-species infection by using intergenotypic chimeric viruses.

Hepatitis E virus (HEV) infects humans and more than a dozen other animal species. We previously showed that open reading frame 2 (ORF2) and ORF3 are apparently not involved in HEV cross-species infection, which infers that the ORF1 may contribute to host tropism. In this study, we utilize the genomic backbone of HEV-1 which only infects humans to construct a panel of intergenotypic chimeras in which the entire ORF1 gene or its functional domains were swapped with the corresponding regions from HEV-3 that infects both humans and pigs. We demonstrated that the chimeric HEVs were replication competent in human liver cells. Subsequently, we intrahepatically inoculated the RNA transcripts of chimeras into pigs to determine if the swapped ORF1 regions confer the chimeras' ability to infect pigs. We showed that there was no evidence of infectivity in pigs for any of the chimeras. We also investigated the role of human ribosome protein sequence S17, which expanded host range in cultured cells, in HEV cross-species infection. We demonstrated that S17 insertion in HEV ORF1 did not abolish HEV replication competency in vitro, but also did not expand HEV host tropism in vivo. The results highlight the complexity of the underlying mechanism of HEV cross-species infection.

Association between thyroid nodule size and malignancy rate.

INTRODUCTION: The diagnostic performance of ultrasound-fine needle aspiration to identify thyroid nodules harbouring malignancy remains variable. The aim of this study was to determine thyroid nodule size and cytological classification as predictors of malignancy risk. MATERIALS AND METHODS: We conducted a retrospective cohort analysis at an academic hospital involving 499 consecutive patients who underwent thyroid surgery between 2004 and 2015. RESULTS: A total of 503 thyroid nodules (499 patients, 84% female; mean age 50.8 years, standard deviation, SD, 15.4 years) were analysed. Of these, 19.5% were malignant. The mean (± SD) nodule size was 3.28 ± 1.63 cm and 3.27 ± 1.54 cm for benign and malignant nodules, respectively. The odds of malignancy for thyroid nodules less than 3.0 cm was similar to those for nodules of 3.0 cm or greater (0.26 compared with 0.29; p=0.77). Overall, the sensitivity and specificity of fine-needle aspiration in this cohort were 71.4% and 100%, respectively. The overall false negative rate was 5.4%. When the cut-off of 3.0 cm was used, the false negative rate in thyroid nodules less than 3.0 cm was 0% compared with 7.0% in nodules of 3.0 cm or greater. Thus, class (p<0.01) but not nodule size (p=0.49), was associated with higher malignancy risk. CONCLUSIONS: Our results suggest that thyroid nodule size did not accurately predict the risk of thyroid malignancy irrespective of fine-needle aspiration cytology. Routine diagnostic thyroid lobectomy solely owing to thyroid nodule size of 3.0 cm or greater is currently not justified.

Cytotoxic T lymphocyte epitopes identified from a contemporary strain of porcine reproductive and respiratory syndrome virus enhance CD4+CD8+ T, CD8+ T, and γδ T cell responses.

Immuno-stimulatory class I-restricted cytotoxic T lymphocytes (CTL) epitopes of porcine reproductive and respiratory syndrome virus (PRRSV) are important for vaccine development. In this study we first determined the expression frequency of swine leukocyte antigen (SLA) class I alleles in commercial pigs in the United States. The SLA genotyping result allowed us to predict potential CTL epitopes from a contemporary strain of PRRSV (RFLP 1-7-4) by using bioinformatic tools. The predicted epitopes were then evaluated in an ex vivo stimulation assay with peripheral blood mononuclear cells isolated from pigs experimentally-infected with PRRSV. Using flow-cytometry analysis, we identified a number of immuno-stimulatory CTL epitopes, including two peptides from GP3 and two from Nsp9 that significantly improved both degranulation marker CD107a and IFN-γ production in cytotoxic CD4+CD8+ T cells, CD8+ T cells, and γδ T cells, and two peptides that inhibited IFN-γ production. These CTL epitopes will aid future vaccine development against PRRSV.

Natalizumab induced cutaneous sarcoidosis-like reaction.

We present a case of a drug-induced sarcoidosis -like reaction (DISR) occurring following initiation of Natalizumab for multiple sclerosis. The reaction was purely cutaneous, and disappeared following drug withdrawal. We highlight this case to the practicing neurologists, with warning to be wary of a new rash on immunomodulatory therapies.

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.