Langat virus inhibits the gp130/JAK/STAT signaling by reducing the gp130 protein level.

The tick-borne encephalitis virus (TBEV) serocomplex includes several medically important flavivirus members endemic to Europe, Asia, and North America, which can induce severe neuroinvasive or viscerotropic diseases with unclear mechanisms of pathogenesis. Langat virus (LGTV) shares a high sequence identity with TBEV but exhibits lower pathogenic potential in humans and serves as a model for virus-host interactions. In this study, we demonstrated that LGTV infection inhibits the activation of gp130/JAK/STAT (Janus kinases (JAK) and signal transducer and activator of transcription (STAT)) signaling, which plays a pivotal role in numerous biological processes. Our data show that the LGTV-infected cells had significantly lower phosphorylated STAT3 (pSTAT3) protein upon oncostatin M (OSM) stimulation than the mock-infected control. LGTV infection blocked the nuclear translocation of STAT3 without a significant effect on total STAT3 protein level. LGTV inhibited JAK1 activation and reduced gp130 protein expression in infected cells, with the viral NS5 protein mediating this effect. TBEV infection also reduces gp130 level. On the other hand, pretreatment of Vero cells with OSM significantly reduces LGTV replication, and STAT1/STAT2 knockdown had little effect on OSM-mediated antiviral effect, which suggests it is independent of STAT1/STAT2 and, instead, it is potentially mediated by STAT3 signlaing. These findings shed light on the LGTV and TBEV-cell interactions, offering insights for the future development of antiviral therapeutics and improved vaccines.

Nonstructural Protein 11 of Porcine Reproductive and Respiratory Syndrome Virus Induces STAT2 Degradation To Inhibit Interferon Signaling.

Interferons (IFNs) play a crucial role in host antiviral response by activating the JAK/STAT (Janus kinase/signal transducer and activator of transcription) signaling pathway to induce the expression of myriad genes. STAT2 is a key player in the IFN-activated JAK/STAT signaling. Porcine reproductive and respiratory syndrome virus (PRRSV) is an important viral pathogen, causing huge losses to the swine industry. PRRSV infection elicits a meager protective immune response in pigs. The objective of this study was to investigate the effect of PRRSV on STAT2 signaling. Here, we demonstrated that PRRSV downregulated STAT2 to inhibit IFN-activated signaling. PRRSV strains of both PRRSV-1 and PRRSV-2 species reduced the STAT2 protein level, whereas the STAT2 transcript level had minimal change. PRRSV reduced the STAT2 level in a dose-dependent manner and shortened STAT2 half-life significantly from approximately 30 to 5 h. PRRSV-induced STAT2 degradation could be restored by treatment with the proteasome inhibitor MG132 and lactacystin. In addition, PRRSV nonstructural protein 11 (nsp11) was identified to interact with and reduce STAT2. The N-terminal domain (NTD) of nsp11 was responsible for STAT2 degradation and interacted with STAT2 NTD and the coiled-coil domain. Mutagenesis analysis showed that the amino acid residue K59 of nsp11 was indispensable for inducing STAT2 reduction. Mutant PRRSV with the K59A mutation generated by reverse genetics almost lost the ability to reduce STAT2. Together, these results demonstrate that PRRSV nsp11 antagonizes IFN signaling via mediating STAT2 degradation and provide further insights into the PRRSV interference of the innate immunity.IMPORTANCE PRRSV infection elicits a meager protective immune response in pigs. One of the possible reasons is that PRRSV antagonizes interferon induction and its downstream signaling. Interferons are key components in the innate immunity and play crucial roles against viral infection and in the activation of adaptive immune response via JAK/STAT signaling. STAT2 is indispensable in the JAK/STAT signaling since it is also involved in activation of antiviral activity in the absence of STAT1. Here, we discovered that PRRSV nsp11 downregulates STAT2. Interestingly, the N-terminal domain of nsp11 is responsible for inducing STAT2 degradation and directly interacts with STAT2 N-terminal domain. We also identified a crucial amino acid residue K59 in nsp11 since a mutation of it led to loss of the ability to downregulate STAT2. A mutant PRRSV with mutation of K59 had minimal effect on STAT2 reduction. Our data provide further insights into PRRSV interference with interferon signaling.

Dexmedetomidine alleviates smoke-induced bronchial and alveolar epithelial cell injury.

Dexmedetomidine (Dex) is a selective α2-adrenoceptor agonist and has ability to prevent inflammation and apoptosis in tissues injury. However, whether Dex could alleviate smoke-induced lung injury remains unknown. This study aimed to explore the protective effects of Dex against smoke-induced lung injury. Bronchial and alveolar epithelial cells were treated with cigarette smoke extract (CSE) for 24 h to simulate cigarette smoke-induced lung injury. Results showed that CSE reduced cell viability and increased levels of pro-inflammatory cytokines TNFα, IL-1ß and IL-6, thus activating NF-κB and COX2 expression. CSE also increased ROS generation, whereas lessened MnSOD and catalase generation. Besides, the ratio of apoptotic cells was enhanced upon CSE stimuli, together with disturbance of apoptotic-related proteins including Bcl-2, Bax and caspase-3. However, Dex reduced the damage of CSE to cell viability. The increased activities of TNFα, IL-1ß and IL-6 induced by CSE were partially attenuated by Dex. Dex also recovered the levels of NF-κB and COX2, as well as mnSOD, catalase and ROS. Furthermore, the increase of cell apoptosis together with imbalance of apoptotic proteins induced by CSE was rescued by Dex. Our results demonstrated that Dex alleviated CSE-induced lung injury through inhibition of inflammation, oxidative stress and apoptosis.

Karyopherin Alpha 6 Is Required for Replication of Porcine Reproductive and Respiratory Syndrome Virus and Zika Virus.

Movement of macromolecules between the cytoplasm and the nucleus occurs through the nuclear pore complex (NPC). Karyopherins comprise a family of soluble transport factors facilitating the nucleocytoplasmic translocation of proteins through the NPC. In this study, we found that karyopherin α6 (KPNA6; also known as importin α7) was required for the optimal replication of porcine reproductive and respiratory syndrome virus (PRRSV) and Zika virus (ZIKV), which are positive-sense, single-stranded RNA viruses replicating in the cytoplasm. The KPNA6 protein level in virus-infected cells was much higher than that in mock-infected controls, whereas the KPNA6 transcript remains stable. Viral infection blocked the ubiquitin-proteasomal degradation of KPNA6, which led to an extension of the KPNA6 half-life and the elevation of the KPNA6 level in comparison to mock-infected cells. PRRSV nsp12 protein induced KPNA6 stabilization. KPNA6 silencing was detrimental to the replication of PRRSV, and KPNA6 knockout impaired ZIKV replication. Moreover, KPNA6 knockout blocked the nuclear translocation of PRRSV nsp1ß but had a minimal effect on two other PRRSV proteins with nuclear localization. Exogenous restitution of KPNA6 expression in the KPNA6-knockout cells results in restoration of the nuclear translocation of PRRSV nsp1ß and the replication of ZIKV. These results indicate that KPNA6 is an important cellular factor for the replication of PRRSV and ZIKV.IMPORTANCE Positive-sense, single-stranded RNA (+ssRNA) viruses replicate in the cytoplasm of infected cells. The roles of transport factors in the nucleocytoplasmic trafficking system for the replication of +ssRNA viruses are not known. In this study, we discovered that PRRSV and ZIKV viruses needed karyopherin α6 (KPNA6), one of the transport factors, to enhance the virus replication. Our data showed that viral infection induced an elevation of the KPNA6 protein level due to an extension of the KPNA6 half-life via viral interference of the ubiquitin-proteasomal degradation of KPNA6. Notably, KPNA6 silencing or knockout dramatically reduced the replication of PRRSV and ZIKV. PRRSV nsp1ß depended on KPNA6 to translocate into the nucleus. In addition, exogenous restitution of KPNA6 expression in KPNA6-knockout cells led to the restoration of nsp1ß nuclear translocation and ZIKV replication. These results reveal a new aspect in the virus-cell interaction and may facilitate the development of novel antiviral therapeutics.

A novel method to rescue and culture duck Astrovirus type 1 in vitro.

BACKGROUND: Reverse genetics systems enable the manipulation of viral genomes and therefore serve as robust reverse genetic tools to study RNA viruses. A DNA-launched rescue system initiates the transcription of viral genomic cDNA from eukaryotic promoter in transfected cells, generating homogenous RNA transcripts in vitro and thus enhancing virus rescue efficiency. As one of the hazardous pathogens to ducklings, the current knowledge of the pathogenesis of duck astrovirus type 1 (DAstV-1) is limited. The construction of a DNA-launched rescue system can help to accelerate the study of the virus pathogenesis. However, there is no report of such a system for DAstV-1. METHODS: In this study, a DNA-launched infectious clone of DAstV-1 was constructed from a cDNA plasmid, which contains a viral cDNA sequence flanked by hammerhead ribozyme (HamRz) and a hepatitis delta virus ribozyme (HdvRz) sequence at both terminals of the viral genome. A silent nucleotide mutation creating a Bgl II site in the ORF2 gene was made to distinguish the rescued virus (rDAstV-1) from the parental virus (pDAstV-1). Immunofluorescence assay (IFA) and western blot were conducted for rescued virus identification in duck embryo fibroblast (DEF) cells pre-treated with trypsin. The growth characteristics of rDAstV-1 and pDAstV-1 in DEF cells and the tissue tropism in 2-day-old ducklings of rDAstV-1 and pDAstV-1 were determined. RESULTS: The infectious DAstV-1 was successfully rescued from baby hamster kidney (BHK-21) cells and could propagate in DEF cells pre-treated with 1 µg/ml trypsin. Upon infection of DEF cells pre-treated with trypsin, DAstV-1 mRNA copies were identified after serial passaging, and the result showed that rDAstV-1 and pDAstV-1 shared similar replication kinetics. Animal experiment showed that the rDAstV-1 had an extensive tissue tropism, and the virus was capable of invading both the central and the peripheral immune organs in infected ducklings. CONCLUSIONS: An improved DNA-launched reverse genetics system for DAstV-1 was firstly constructed. Infectious virus recovered from BHK-21 cells could propagate in DEF cells pre-treated with trypsin. This is the first report of the successful in vitro cultivation of DAstV-1. We believe this valuable experimental system will contribute to the further study of DAstV-1 genome function and pathogenesis.

Quantitative Proteomic Analysis Uncovers the Mediation of Endoplasmic Reticulum Stress-Induced Autophagy in DHAV-1-Infected DEF Cells.

Autophagy is a tightly regulated catabolic process and is activated in cells in response to stress signals. Despite extensive study, the interplay between duck hepatitis A virus type 1 (DHAV-1) and the autophagy of host cells is not clear. In this study, we applied proteomics analysis to investigate the interaction mechanism between DHAV-1 and duck embryo fibroblast (DEF) cells. In total, 507 differentially expressed proteins (DEPs) were identified, with 171 upregulated proteins and 336 downregulated proteins. The protein expression level of heat shock proteins (Hsps) and their response to stimulus proteins and zinc finger proteins (ZFPs) were significantly increased while the same aspects of ribosome proteins declined. Bioinformatics analysis indicated that DEPs were mainly involved in the "response to stimulus", the "defense response to virus", and the "phagosome pathway". Furthermore, Western blot results showed that the conversion of microtubule-associated protein 1 light chain 3-I (LC3-I) to the lipidation form of LC3-II increased, and the conversion rate decreased when DEF cells were processed with 4-phenylbutyrate (4-PBA). These findings indicated that DHAV-1 infection could cause endoplasmic reticulum (ER) stress-induced autophagy in DEF cells, and that ER stress was an important regulatory factor in the activation of autophagy. Our data provide a new clue regarding the host cell response to DHAV-1 and identify proteins involved in the DHAV-1 infection process or the ER stress-induced autophagy process.

Porcine Reproductive and Respiratory Syndrome Virus Antagonizes JAK/STAT3 Signaling via nsp5, Which Induces STAT3 Degradation.

Signal transducer and activator of transcription 3 (STAT3) is a pleiotropic signaling mediator of many cytokines, including interleukin-6 (IL-6) and IL-10. STAT3 is known to play critical roles in cell growth, proliferation, differentiation, immunity and inflammatory responses. The objective of this study was to determine the effect of porcine reproductive and respiratory syndrome virus (PRRSV) infection on the STAT3 signaling since PRRSV induces a weak protective immune response in host animals. We report here that PRRSV infection of MARC-145 cells and primary porcine pulmonary alveolar macrophages led to significant reduction of STAT3 protein level. Several strains of both PRRSV type 1 and type 2 led to a similar reduction of STAT3 protein level but had a minimal effect on its transcripts. The PRRSV-mediated STAT3 reduction was in a dose-dependent manner as the STAT3 level decreased, along with incremental amounts of PRRSV inocula. Further study showed that nonstructural protein 5 (nsp5) of PRRSV induced the STAT3 degradation by increasing its polyubiquitination level and shortening its half-life from 24 h to ∼3.5 h. The C-terminal domain of nsp5 was shown to be required for the STAT3 degradation. Moreover, the STAT3 signaling in the cells transfected with nsp5 plasmid was significantly inhibited. These results indicate that PRRSV antagonizes the STAT3 signaling by accelerating STAT3 degradation via the ubiquitin-proteasomal pathway. This study provides insight into the PRRSV interference with the JAK/STAT3 signaling, leading to perturbation of the host innate and adaptive immune responses. IMPORTANCE: The typical features of immune responses in PRRSV-infected pigs are delayed onset and low levels of virus neutralizing antibodies, as well as weak cell-mediated immunity. Lymphocyte development and differentiation rely on cytokines, many of which signal through the JAK/STAT signaling pathway to exert their biological effects. Here, we discovered that PRRSV antagonizes the JAK/STAT3 signaling by inducing degradation of STAT3, a master transcription activator involved in multiple cellular processes and the host immune responses. The nsp5 protein of PRRSV is responsible for the accelerated STAT3 degradation. The PRRSV-mediated antagonizing STAT3 could lead to suppression of a broad spectrum of cytokines and growth factors to allow virus replication and spread in host animals. This may be one of the reasons for the PRRSV interference with the innate immunity and its poor elicitation of protective immunity. This finding provides insight into PRRSV pathogenesis and its interference with the host immune responses.

Construction and characterization of an improved DNA-launched infectious clone of duck hepatitis a virus type 1.

BACKGROUND: DNA-launched infectious system is a useful tool with high rescue efficiency that allows the introduction of mutations in specific positions to investigate the function of an individual viral element. Rescued virus particles could be harvested by directly transfecting the DNA-launched recombinant plasmid to the host cells, which will reduce labor and experimental cost by skipping the in vitro transcription assay. METHODS: A total of four overlapping fragments covering the entire viral genome were amplified and then were assembled into a transformation vector based on pIRES2-EGFP to establish the DNA-launched infectious system of duck hepatitis A virus type 1 (DHAV-1), named pIR-DHAV-1. Reverse transcription polymerase chain reaction (RT-PCR) detection, quantitative real-time polymerase chain reaction (qRT-PCR), western blotting assay and indirect immunofluorescence (IFA) were conducted for rescued virus identification. A total of 4.0 µg of recombinant plasmid of pIR-DHAV-1 and in vitro transcribed product of 4.0 µg of RNA-launched infectious clone named pR-DHAV-1 were transfected into BHK-21 cells to analyze the rescue efficiency. Following that, tissue tropism of rescued virus (rDHAV-1) and parental virus (pDHAV-1) were assayed for virulence testing in 1-day-old ducklings. RESULTS: Rescued virus particles carry the designed genetic marker which could be harvested by directly transfecting pIR-DHAV-1 to BHK-21 cells. The qRT-PCR and western blotting results indicated that rDHAV-1 shared similar growth characteristics with pDHAV-1. Furthermore, DNA-launched infectious system possessed much higher rescue efficiency assay compared to RNA-launched infectious system. The mutation at position 3042 from T to C has no impact on viral replication and tissue tropism. From 1 h post infection (hpi) to 48 hpi, the viral RNA copies of rDHAV-1 in liver were the highest among the six tested tissues (with an exception of thymus at 6 hpi), while the viral RNA copy numbers in heart and kidney were alternately the lowest. CONCLUSION: We have constructed a genetically stable and highly pathogenic DNA-launched infectious clone, from which the rescued virus could be harvested by direct transfection with recombinant plasmids. rDHAV-1 shared similar growth characteristics and tissue tropism with pDHAV-1. The DNA-launched infectious system of DHAV-1 possessed higher rescue efficiency compared to the traditional RNA-launched infectious system.

Circulation and in vivo distribution of duck hepatitis A virus types 1 and 3 in infected ducklings.

The circulation of duck hepatitis A virus types 1 (DHAV-1) and 3 (DHAV-3) in Southeast Asia has resulted in a continuously changing epidemiological scenario. In this study, a duplex real-time PCR assay for simultaneous quantitative detection of DHAV-1 and DHAV-3 was established, and 200 liver samples from dead ducklings collected from 31 different flocks in Shandong province, China, were tested. Fifty-eight (29.0 %) samples from 13 flocks were positive for DHAV-1 single infection, 113 (56.5 %) samples from 13 other flocks were positive for DHAV-3 single infection, and 24 samples (12.0 %) from four flocks were positive for both viruses. DHAV-1 and DHAV-3 were detected with high viral loads in all of the organs tested (liver, spleen, pancreas, kidney, heart, thymus, bursa of Fabricius and brain). No significant difference in DHAV-1 and DHAV-3 viral loads was found between singly infected and coinfected samples, and there was no correlation between the viral loads of the two viruses and the age of dead ducklings. To the best of our knowledge, this is the first report about the in vivo distribution of DHAV-1 and DHAV-3 in clinically infected ducklings.

Hepatitis E Virus: Isolation, Propagation, and Quantification.

Hepatitis E virus (HEV) predominantly causes acute liver disease in humans and is transmitted via the fecal-oral route. HEV infection in pregnant women can result in grave consequences, with up to 30% fatality. The HEV strains infecting humans mainly belong to four genotypes. Genotypes 1 and 2 are restricted to human infection, while genotypes 3 and 4 are zoonotic. HEV genotype 3 (HEV-3) can cause both acute and chronic liver disease. Several cell lines (mainly hepatocytes) have been developed for HEV propagation and biological study. However, HEV production in these cell lines is suboptimal and inefficient. Here, we present methods for the isolation, propagation, and quantification of HEV. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Isolation and propagation of hepatitis E virus in cultured cells from clinical HEV specimens Support Protocol 1: Quantification of HEV RNA by RT-qPCR Basic Protocol 2: Recovery of HEV from infectious cDNA clones and purification of the virus Support Protocol 2: Quantification of HEV live particles by infectivity assay.

Creatine phosphate improves myocardial function and myocardial enzyme profile in children with myocarditis.

Myocarditis in children is more common in clinical practice, which can cause different degrees of cardiac function damage. We investigated the effects of creatine phosphate in the treatment of myocarditis in children. Children in the control group were treated with sodium fructose diphosphate, and children in the observation group were treated with creatine phosphate on the basis of the control group. After treatment, the myocardial enzyme profile and cardiac function of children in the observation group were better than the control group. The total effective rate of treatment for children in the observation group was higher than that in the control group. In conclusion, creatine phosphate could significantly improve myocardial function, improve myocardial enzyme profile and reduce myocardial damage in children with pediatric myocarditis and had a high safety of use, which was worthy of clinical promotion.

Zika Virus Induces Degradation of the Numb Protein Required through Embryonic Neurogenesis.

Zika virus (ZIKV) is a mosquito-borne flavivirus and causes an infection associated with congenital Zika syndrome and Guillain-Barre syndrome. The mechanism of ZIKV-mediated neuropathogenesis is not well understood. In this study, we discovered that ZIKV induces degradation of the Numb protein, which plays a crucial role in neurogenesis by allowing asymmetric cell division during embryonic development. Our data show that ZIKV reduced the Numb protein level in a time- and dose-dependent manner. However, ZIKV infection appears to have minimal effect on the Numb transcript. Treatment of ZIKV-infected cells with a proteasome inhibitor restores the Numb protein level, which suggests the involvement of the ubiquitin-proteasome pathway. In addition, ZIKV infection shortens the half-life of the Numb protein. Among the ZIKV proteins, the capsid protein significantly reduces the Numb protein level. Immunoprecipitation of the Numb protein co-precipitates the capsid protein, indicating the interaction between these two proteins. These results provide insights into the ZIKV-cell interaction that might contribute to its impact on neurogenesis.

ZIKV infection differentially affects the transcriptional profiles in HTR8 and U251 cells.

The mechanism by which Zika virus (ZIKV) causes severe birth defects in pregnant women remains unclear. Cell tropisms in placenta and brain play a crucial role in ZIKV pathogenesis, leading to congenital Zika syndrome (CZS). To identify the host factors involved in ZIKV infection, we compared the transcriptional profiles of ZIKV-infected human first-trimester placental trophoblast cells HTR8/SVneo and a human glioblastoma astrocytoma cell line U251. Our results demonstrated that ZIKV exhibited lower rates of mRNA replication and protein expression in HTR8 than in U251 cells, while showing a higher release of infectious viral particles. However, a greater number of differentially expressed genes (DEGs) were found in ZIKV-infected U251 cells than in ZIKV-infected HTR8 cells. Several of these DEGs were enriched in distinct biological processes related to the characteristics of each cell type that may contribute to foetal damage. Both cell types exhibited activation of common interferons, inflammatory cytokines, and chemokine production upon ZIKV infection. Moreover, the neutralization of tumour necrosis factor-alpha (TNF-α) promoted ZIKV infection in both trophoblasts and glioblastoma astrocytoma cells. Overall, we identified multiple DEGs associated with ZIKV pathogenesis.

Immunotherapy for hepatobiliary cancers: Emerging targets and translational advances.

Over the past several decades, primary liver cancer (PLC), mostly hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinoma (iCCA), has become the focus of rising concern mainly due to the increasing rates of incidence and high global mortality. Immunotherapy, as an emerging treatment approach, represents an effective and promising option against PLC. However, the selection of immunotherapeutic targets while considering tumor heterogeneity and immunosuppressive tumor microenvironment is a major challenge. The purpose of this review is to summarize and present the emerging immunotherapeutic targets for HCC and iCCA and to evaluate their translation advances in currently ongoing clinical trials. To better provide a framework for the liver cancer target selection, this chapter will highlight cell surface antigens expressed in both tumor cells and immune cells. Particular focus will be on the development, biology and function of Glypican-3 (GPC3) and Mesothelin (MSLN) in the cancer progress of HCC and iCCA, respectively. By doing so, we will explore the prospects and applications of various immunotherapeutic strategies such as vaccines, monoclonal antibodies, immunotoxins, antibody-drug conjugates (ADCs) and chimeric antigen receptors (CARs) T cells that have been developed targeting GPC3 and MSLN.

Efficacy of Jiedu Pingsou Decoction Combined with Azithromycin in the Treatment of Children with Mycoplasma Pneumonia and Its Effects on Inflammatory Factors and Immune Function.

Objective: The purpose of this study is to detect the clinical efficacy of Jiedu Pingsou Decoction combined with azithromycin in the treatment of children with mycoplasma pneumonia and the effect on inflammatory factors and immune function in children. Methods: A total of 68 children with mycoplasma pneumonia in our hospital from January 2021 to January 2022 were included in this study, and they were randomly divided into the control group and the observation group with 34 cases in each group. The children in the control group were treated with azithromycin, and the children in the observation group were treated with Jiedu Pingsou Decoction on this basis. The clinical manifestations, treatment effects, blood routine, serum inflammatory factor levels, and T cell subsets before and after treatment were compared between the two groups. Results: The total effective rate in the observation group was 94.12%, which was higher than that in the control group, which was 82.35%, and the difference between the two groups was statistically significant (P < 0.05). After treatment, the levels of CD3+, CD4+, and CD4+/CD8+ in the two groups were higher than those before treatment, and the level of CD8+ was lower than before treatment. The difference between groups was statistically significant (P < 0.05). The levels of serum C-reactive protein (CRP), tumor necrosis factor-α (TNF-α), interferon gamma (IFN-γ), interleukin-6 (IL-6), and interleukin-10 (IL-10) in the two groups after treatment were lower than those before treatment, and the difference between the two groups was statistically significant (P < 0.05). The difference between groups was statistically significant (P < 0.05). There were 4 cases and 2 cases of adverse reactions in the control group and the observation group, respectively, and the difference between the two groups was statistically significant (P > 0.05). Conclusion: Jiedu Pingsou Decoction combined with azithromycin can effectively improve the levels of T cell subsets, immune function, and inflammatory factors in children with mycoplasma pneumonia, improve clinical symptoms, and is safe and stable, and can be used in clinical practice.

Advances in Hepatitis E Virus Biology and Pathogenesis.

Hepatitis E virus (HEV) is one of the causative agents for liver inflammation across the world. HEV is a positive-sense single-stranded RNA virus. Human HEV strains mainly belong to four major genotypes in the genus Orthohepevirus A, family Hepeviridae. Among the four genotypes, genotype 1 and 2 are obligate human pathogens, and genotype 3 and 4 cause zoonotic infections. HEV infection with genotype 1 and 2 mainly presents as acute and self-limiting hepatitis in young adults. However, HEV infection of pregnant women with genotype 1 strains can be exacerbated to fulminant hepatitis, resulting in a high rate of case fatality. As pregnant women maintain the balance of maternal-fetal tolerance and effective immunity against invading pathogens, HEV infection with genotype 1 might dysregulate the balance and cause the adverse outcome. Furthermore, HEV infection with genotype 3 can be chronic in immunocompromised patients, with rapid progression, which has been a challenge since it was reported years ago. The virus has a complex interaction with the host cells in downregulating antiviral factors and recruiting elements to generate a conducive environment of replication. The virus-cell interactions at an early stage might determine the consequence of the infection. In this review, advances in HEV virology, viral life cycle, viral interference with the immune response, and the pathogenesis in pregnant women are discussed, and perspectives on these aspects are presented.

A Novel Peptide Isolated from a Phage Display Peptide Library Modeling Antigenic Epitope of DHAV-1 and DHAV-3.

Duck hepatitis A virus (DHAV), the major pathogen of duck virus hepatitis (DVH), causes severe diseases that threaten the duck industry worldwide. The VP1 protein, a major structural protein of DHAV, is able to induce neutralizing antibody in ducks. The purpose of this study was to identify the antigenic mimotope of DHAV by phage display technology. A monoclonal antibody (mAb) 4E6 against DHAV-1 and DHAV-3 was prepared, and a phage library prepared with the PhD-12 Phage Display Peptide Library Kit was screened with the mAb. A novel peptide, 1GLTWKLPPSM10 was identified with high affinity to the mAb and could specifically block mAb 4E6 from binding DHAV-1 and DHAV-3. Animal tests confirmed that the immunization of ducklings with the mimotope could inhibit the virus proliferation and protect the ducklings from DVH. In summary, the neutralizing conformational mimotope 1GLTWKLPPSM10 might be a promising vaccine candidate for the prevention of DHAV infection.

Zika virus NS2A protein induces the degradation of KPNA2 (karyopherin subunit alpha 2) via chaperone-mediated autophagy.

KPNA2/importin-alpha1 (karyopherin subunit alpha 2) is the primary nucleocytoplasmic transporter for some transcription factors to activate cellular proliferation and differentiation. Aberrant increase of KPNA2 level is identified as a prognostic marker in a variety of cancers. Yet, the turnover mechanism of KPNA2 remains unknown. Here, we demonstrate that KPNA2 is degraded via the chaperone-mediated autophagy (CMA) and that Zika virus (ZIKV) enhances the KPNA2 degradation. KPNA2 contains a CMA motif, which possesses an indispensable residue Gln109 for the CMA-mediated degradation. RNAi-mediated knockdown of LAMP2A, a vital component of the CMA pathway, led to a higher level of KPNA2. Moreover, ZIKV reduced KPNA2 via the viral NS2A protein, which contains an essential residue Thr100 for inducing the CMA-mediated KPNA2 degradation. Notably, mutant ZIKV with T100A alteration in NS2A replicates much weaker than the wild-type virus. Also, knockdown of KPNA2 led to a higher ZIKV viral yield, which indicates that KPNA2 mediates certain antiviral effects. These data provide insights into the KPNA2 turnover and the ZIKV-cell interactions.

gga-microRNA-375 negatively regulates the cell cycle and proliferation by targeting Yes-associated protein 1 in DF-1 cells.

MicroRNAs (miRNAs/miRs) serve a key role in regulating the cell cycle and inducing tumorigenesis. Subgroup J of the avian leukosis virus (ALV-J) belongs to the family Retroviridae, subfamily Orthoretrovirinae and genus Alpharetrovirus that causes tumors in susceptible chickens. gga-miR-375 is downregulated and Yes-associated protein 1 (YAP1) is upregulated in ALV-J-induced tumors in the livers of chickens, and it has been further identified that YAP1 is the direct target gene of gga-miR-375. In the present study, it was found that ALV-J infection promoted the cell cycle and proliferation in DF-1 cells. As the cell cycle and cell proliferation are closely associated with tumorigenesis, further experiments were performed to determine whether gga-miR-375 and YAP1 were involved in these cellular processes. It was demonstrated that gga-miR-375 significantly inhibited the cell cycle by inhibiting G1 to S/G2 stage transition and decreasing cell proliferation, while YAP1 significantly promoted the cell cycle and proliferation. Furthermore, these cellular processes in DF-1 cells were affected by gga-miR-375 through the targeting of YAP1. Collectively, the present results suggested that gga-miR-375, downregulated by ALV-J infection, negatively regulated the cell cycle and proliferation via the targeting of YAP1.

Recombinant Lactobacillus acidophilus expressing S1 and S2 domains of porcine epidemic diarrhea virus could improve the humoral and mucosal immune levels in mice and sows inoculated orally.

Porcine epidemic diarrhea (PED) is a highly contagious intestinal infectious disease caused by porcine epidemic diarrhea virus (PEDV), which is characterized by a high mortality rate in piglets. Since 2012, a remarkable growth in PED outbreaks occurred in many pig farms in China, landing a heavy blow on the pig industry. In order to develop a new effective vaccine for the current PEDV, oral vaccines were generated by transferring eukaryotic expression recombinant plasmids carrying the S1 and S2 (antigenic sites of the S protein) epitopes of PEDV into a swine-origin Lactobacillus acidophilus (L. acidophilus). After oral immunization of the BALB/c mice, higher levels of anti-PEDV specific IgG and SIgA antibodies and cellular immune responses were detected in mice orally administered with the recombinant L. acidophilus-S1 compared to the L. acidophilus-S2. Furthermore, L. acidophilus-S1 was used to inoculate the pregnant sows orally and the results showed that the recombinant L. acidophilus-S1 could elicit a specific systemic and mucosal immune response. In summary, our study demonstrated that oral immunization with L. acidophilus-S1 could improve the humoral and mucosal immune levels in sows and would be a promising candidate vaccine against PEDV infection in piglets.