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In vitro inhibition of porcine hemagglutinating encephalomyelitis virus replication with siRNAs targeting the spike glycoprotein and replicase polyprotein genes.

ABSTRACT: OBJECTIVE:The specific effect of rna interference on the replication of porcine hemagglutinating encephalomyelitis virus (phe-cov) was explored. METHODS:Four species of small interfering RNA (siRNA), targeting different regions of the PHE-CoV spike glycoprotein and replicase polyprotein genes, were prepared by in vitro transcription. After transfection of PK-15 cells with each of the siRNAs followed by infection with PHE-CoV, the cytopathic effect (CPE) was examined by phase-contrast microscope, and viral proliferation within cells was examined by indirect immunofluorescence microscopy, hemagglutination (HA) test, TCID(50) assay and real-time RT-PCR. RESULTS:Examination of CPE demonstrated that the four siRNAs were capable of protecting cells against PHE-CoV invasion with very high specificity and efficiency. At 48 h post-infection, only a few siRNA-treated cells were positive for viral antigen staining, whereas most untreated virus-infected cells were positive. Transfection with siRNAs also suppressed the production of infectious virus by up to 18- to 32-fold as assessed by a HA test and 93- to 494-fold as assessed by TCID(50) assay. Furthermore, treatment with siRNAs caused a 53-91% reduction in the viral genome copy number as assessed by real-time RT-PCR. CONCLUSION:These results suggested that the four species of siRNAs can efficiently inhibit PHE-CoV genome replication and infectious virus production.

SUBMITTER: Lan Y

PROVIDER: S-EPMC7179546 | biostudies-literature | 2012

REPOSITORIES: biostudies-literature

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In vitro inhibition of porcine hemagglutinating encephalomyelitis virus replication with siRNAs targeting the spike glycoprotein and replicase polyprotein genes.

Lan Yungang Y Lu Huijun H Zhao Kui K He Wenqi W Chen Keyan K Wang Gaili G Song Deguang D Gao Feng F

Intervirology 20110303 1

<h4>Objective</h4>The specific effect of rna interference on the replication of porcine hemagglutinating encephalomyelitis virus (phe-cov) was explored.<h4>Methods</h4>Four species of small interfering RNA (siRNA), targeting different regions of the PHE-CoV spike glycoprotein and replicase polyprotein genes, were prepared by in vitro transcription. After transfection of PK-15 cells with each of the siRNAs followed by infection with PHE-CoV, the cytopathic effect (CPE) was examined by phase-contr ...[more]

PMID: 21372550

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Project description:Porcine hemagglutinating encephalomyelitis virus (PHEV), a member of the genus Betacoronavirus, is the causative agent of neurological disease in pigs. No effective therapeutics are currently available for PHEV infection. Resveratrol has been shown to exert neuroprotective and antiviral effects. Here resveratrol was investigated for its ability to inhibit PHEV replication in nerve cells and central nervous system tissues. Anti-PHEV effect of resveratrol was evaluated using an in vitro cell-based PHEV infection model and employing a mouse PHEV infection model. The collected cells or tissues were used for quantitative PCR analysis, western blot analysis, or indirect immunofluorescence assay. The supernatants were collected to quantify viral loads by TCID50 assay in vitro. EC50 and CC50 were determined by dose-response experiments, and the ratio (EC50/CC50) was used as a selectivity index (SI) to measure the antiviral versus cytotoxic activity. Our results showed that resveratrol treatment reduced PHEV titer in a dose-dependent manner, with a 50% inhibition concentration of 6.24 μM. A reduction of > 70% of viral protein expression and mRNA copy number and a 19-fold reduction of virus titer were achieved when infected cells were treated with 10 µM resveratrol in a pre-treatment assay. Quantitative PCR analysis and TCID50 assay results revealed that the addition of 10 μM resveratrol to cells after adsorption of PHEV significantly reduced 56% PHEV mRNA copy number and eightfold virus titer. 10 µM resveratrol treatment reduced 46% PHEV mRNA copy number and fourfold virus titer in virus inactivation assay. Moreover, the in vivo data obtained in this work also demonstrated that resveratrol inhibited PHEV replication, and anti-PHEV activities of resveratrol treatment via intranasal installation displayed better than oral gavage. These results indicated that resveratrol exerted antiviral effects under various drug treatment and virus infection conditions in vitro and holds promise as a treatment for PHEV infection in vivo.

Project description:Porcine hemagglutinating encephalomyelitis virus (PHEV) is a betacoronavirus that causes vomiting and wasting disease and/or encephalomyelitis in suckling pigs. This study characterized PHEV infection, pathogenesis, and immune response in cesarean-derived, colostrum-deprived (CDCD) neonatal pigs. Infected animals developed mild respiratory, enteric, and neurological clinical signs between 2 to 13 days postoronasal inoculation (dpi). PHEV did not produce viremia, but virus shedding was detected in nasal secretions (1 to 10 dpi) and feces (2 to 7 dpi) by reverse transcriptase quantitative PCR (RT-qPCR). Viral RNA was detected in all tissues except liver, but the detection rate and RT-qPCR threshold cycle (CT ) values decreased over time. The highest concentration of virus was detected in inoculated piglets necropsied at 5 dpi in turbinate and trachea, followed by tonsils, lungs, tracheobronchial lymph nodes, and stomach. The most representative microscopic lesions were gastritis lymphoplasmacytic, moderate, multifocal, with perivasculitis, and neuritis with ganglia degeneration. A moderate inflammatory response, characterized by increased levels of interferon alpha (IFN-α) in plasma (5 dpi) and infiltration of T lymphocytes and macrophages were also observed. Increased plasma levels of interleukin-8 (IL-8) were detected at 10 and 15 dpi, coinciding with the progressive resolution of the infection. Moreover, a robust antibody response was detected by 10 dpi. An ex vivo air-liquid CDCD-derived porcine respiratory cells culture (ALI-PRECs) system showed virus replication in ALI-PRECs and cytopathic changes and disruption of ciliated columnar epithelia, thereby confirming the tracheal epithelia as a primary site of infection for PHEV.IMPORTANCE Among the ∼46 virus species in the family Coronaviridae, many of which are important pathogens of humans and 6 of which are commonly found in pigs, porcine hemagglutinating encephalomyelitis remains one of the least researched. The present study provided a comprehensive characterization of the PHEV infection process and immune responses using CDCD neonatal pigs. Moreover, we used an ex vivo ALI-PRECs system resembling the epithelial lining of the tracheobronchial region of the porcine respiratory tract to demonstrate that the upper respiratory tract is a primary site of PHEV infection. This study provides a platform for further multidisciplinary studies of coronavirus infections.

Project description:Members of family Coronaviridae cause a variety of diseases in birds and mammals. Porcine hemagglutinating encephalomyelitis virus (PHEV), a lesser-researched coronavirus, can infect naive pigs of any age, but clinical disease is observed in pigs ≤4 weeks of age. No commercial PHEV vaccines are available, and neonatal protection from PHEV-associated disease is presumably dependent on lactogenic immunity. Although subclinical PHEV infections are thought to be common, PHEV ecology in commercial swine herds is unknown. To begin to address this gap in knowledge, a serum IgG antibody enzyme-linked immunosorbent assay (ELISA) based on the S1 protein was developed and evaluated on known-status samples and then used to estimate PHEV seroprevalence in U.S. sow herds. Assessment of the diagnostic performance of the PHEV S1 ELISA using serum samples (n = 924) collected from 7-week-old pigs (n = 84; 12 pigs per group) inoculated with PHEV, porcine epidemic diarrhea virus, transmissible gastroenteritis virus, porcine respiratory coronavirus, or porcine deltacoronavirus showed that a sample-to-positive cutoff value of ≥0.6 was both sensitive and specific, i.e., all PHEV-inoculated pigs were seropositive from days postinoculation 10 to 42, and no cross-reactivity was observed in samples from other groups. The PHEV S1 ELISA was then used to estimate PHEV seroprevalence in U.S. sow herds (19 states) using 2,756 serum samples from breeding females (>28 weeks old) on commercial farms (n = 104) with no history of PHEV-associated disease. The overall seroprevalence was 53.35% (confidence interval [CI], ±1.86%) and herd seroprevalence was 96.15% (CI, ±3.70%).IMPORTANCE There is a paucity of information concerning the ecology of porcine hemagglutinating encephalomyelitis virus (PHEV) in commercial swine herds. This study provided evidence that PHEV infection is endemic and highly prevalent in U.S. swine herds. These results raised questions for future studies regarding the impact of endemic PHEV on swine health and the mechanisms by which this virus circulates in endemically infected populations. Regardless, the availability of the validated PHEV S1 enzyme-linked immunosorbent assay (ELISA) provides the means for swine producers to detect and monitor PHEV infections, confirm prior exposure to the virus, and to evaluate the immune status of breeding herds.

Project description:Uncoordinated 51-like kinase 1 (ULK1) is a well-characterized initiator of canonical autophagy under basal or pathological conditions. Porcine hemagglutinating encephalomyelitis virus (PHEV), a neurotropic betacoronavirus (β-CoV), impairs ULK1 kinase but hijacks autophagy to facilitate viral proliferation. However, the machinery of PHEV-induced autophagy initiation upon ULK1 kinase deficiency remains unclear. Here, the time course of PHEV infection showed a significant accumulation of autophagosomes (APs) in nerve cells in vivo and in vitro. Utilizing ULK1-knockout neuroblastoma cells, we have identified that ULK1 is not essential for productive AP formation induced by PHEV. In vitro phosphorylation studies discovered that mTORC1-regulated ULK1 activation stalls during PHEV infection, whereas AP biogenesis was controlled by AMPK-driven BECN1 phosphorylation. A lack of BECN1 is sufficient to block LC3 lipidation and disrupt recruitment of the LC3-ATG14 complex. Moreover, BECN1 acts as a bona fide substrate for ULK1-independent neural autophagy, and ectopic expression of BECN1 somewhat enhances PHEV replication. These findings highlight a novel machinery of noncanonical autophagy independent of ULK1 that bypasses the conserved initiation circuit of AMPK-mTORC1-ULK1, providing new insights into the interplay between neurotropic β-CoV and the host. IMPORTANCE The ongoing coronavirus disease 2019 (COVID-19) pandemic alongside the outbreaks of severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome (MERS) pose Betacoronavirus (β-CoV) as a global public health challenge. Coronaviruses subvert, hijack, or utilize autophagy to promote proliferation, and thus, exploring the cross talk between β-CoV and autophagy is of great significance in confronting future β-CoV outbreaks. Porcine hemagglutinating encephalomyelitis virus (PHEV) is a highly neurotropic β-CoV that invades the central nervous system (CNS) in pigs, but understanding of the pathogenesis for PHEV-induced neurological dysfunction is yet limited. Here, we discovered a novel regulatory principle of neural autophagy initiation during PHEV infection, where productive autophagosome (AP) biogenesis bypasses the multifaceted regulation of ULK1 kinase. The PHEV-triggered noncanonical autophagy underscores the complex interactions of virus and host and will help in the development of therapeutic strategies targeting noncanonical autophagy to treat β-CoV disease.

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In vitro inhibition of porcine hemagglutinating encephalomyelitis virus replication with siRNAs targeting the spike glycoprotein and replicase polyprotein genes.

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In vitro inhibition of porcine hemagglutinating encephalomyelitis virus replication with siRNAs targeting the spike glycoprotein and replicase polyprotein genes.

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