Porcine Hemagglutinating Encephalomyelitis Virus Co-Opts Multivesicular-Derived Exosomes for Transmission.

Porcine hemagglutinating encephalomyelitis virus (PHEV) is a member of the family Coronaviridae, genus Betacoronavirus, and subgenus Embecovirus that causes neurological disorders, vomiting and wasting disease (VWD), or influenza-like illness (ILI) in pigs. Exosomes regulate nearby or distant cells as a means of intercellular communication; however, whether they are involved in the transmission of viral reference materials during PHEV infection is unknown. Here, we collected exosomes derived from PHEV-infected neural cells (PHEV-exos) and validated their morphological, structural, and content characteristics. High-resolution mass spectrometry indicated that PHEV-exos carry a variety of cargoes, including host innate immunity sensors and viral ingredients. Furthermore, transwell analysis revealed that viral ingredients, such as proteins and RNA fragments, could be encapsulated in the exosomes of multivesicular bodies (MVBs) to nonpermissive microglia. Inhibition of exosome secretion could suppress PHEV infection. Therefore, we concluded that the mode of infectious transmission of PHEV is likely through a mixture of virus-modified exosomes and virions and that exosomal export acts as a host strategy to induce an innate response in replicating nonpermissive bystander cells free of immune system recognition. IMPORTANCE The novel coronavirus severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a large number of deaths worldwide. Clinical neurological complications have occurred in some cases; however, knowledge of the natural history of coronavirus in the central nervous system (CNS) is thus far limited. PHEV is a typical neurotropic betacoronavirus (ß-CoV) that propagates via neural circuits in the host CNS after peripheral incubation rather than through the bloodstream. It is therefore a good prototype pathogen to investigate the neuropathological pathogenesis of acute human coronavirus infection. In this study, we demonstrate a new association between host vesicle-based secretion and PHEV infection, showing that multivesicular-derived exosomes are one of the modes of infectious transmission and that they mediate the transfer of immunostimulatory cargo to uninfected neuroimmune cells. These findings provide novel insights into the treatment and monitoring of neurological consequences associated with ß-CoV, similar to those associated with SARS-CoV-2.

Suppression of porcine hemagglutinating encephalomyelitis virus replication by resveratrol.

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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.

Complete genome sequence of a novel porcine hemagglutinating encephalomyelitis virus strain identified in South Korea.

Porcine hemagglutinating encephalomyelitis virus (PHEV) is a member of the subgenus Embecovirus of the genus Betacoronavirus, and it is ubiquitously distributed in most pig-farming countries worldwide with low clinical incidence. Here, we report the full-length genome sequence and molecular characterization of a novel PHEV strain identified in diarrheic neonates in South Korea. The complete genome of the Korean PHEV strain GNU-2113 was sequenced and analyzed to characterize PHEV circulating in South Korea. The GNU-2113 genome was determined to be 29,982 nucleotides in length, with large unique deletions in the regions encoding nonstructural protein 3 and NS2. It was found to share 95.1-96.9% sequence identity with other global strains. Genetic and phylogenetic analysis indicated that the GNU-2113 strain is distantly related to the existing PHEV genotypes, implying that the virus appears to undergo substantial evolution under endemic pressure. This study provides important information about the genetic diversity of PHEV circulating subclinically in swine herds, which may ensure viral fitness in the enzootic environment.

Porcine haemagglutinating encephalomyelitis virus deactivates transcription factor IRF3 and limits type I interferon production.

Porcine haemagglutinating encephalomyelitis virus (PHEV) is a member of coronavirus that causes acute infectious disease and high mortality in piglets. The transcription factor IRF3 is a central regulator of type I interferon (IFN) innate immune signalling. Here, we report that PHEV infection of RAW264.7 cells results in strong suppression of IFN-ß production in the early stage. A comparative analysis of the upstream effector of IFN-ß transcription demonstrated that deactivation of IRF3, but not p65 or ATF-2 proteins, is uniquely attributed to failure of early IFN-ß induction. Moreover, the RIG-I/MDA5/MAVS/TBK1-dependent protective response that regulates the IRF3 pathway is not disrupted by PHEV and works well underlying the deactivated IRF3-mediated IFN-ß inhibition. After challenge with poly(I:C), a synthetic analogue of dsRNA used to stimulate IFN-ß secretion in the TLR-controlled pathway, we show that PHEV and poly(I:C) regulate IFN-ß-induction via two different pathways. Collectively, our findings reveal that deactivation of IRF3 is a specific mechanism that contributes to termination of type I IFN signalling during early infection with PHEV independent of the conserved RIG-I/MAVS/MDA5/TBK1-mediated innate immune response.

miR-142a-3p promotes the proliferation of porcine hemagglutinating encephalomyelitis virus by targeting Rab3a.

Porcine hemagglutinating encephalomyelitis virus (PHEV) is a typical neurotropic coronavirus that mainly invades the central nervous system (CNS) in piglets and causes vomiting and wasting disease. Emerging evidence suggests that PHEV alters microRNA (miRNA) expression profiles, and miRNA has also been postulated to be involved in its pathogenesis, but the mechanisms underlying this process have not been fully explored. In this study, we found that PHEV infection upregulates miR-142a-3p RNA expression in N2a cells and in the CNS of mice. Downregulation of miR-142a-3p by an miRNA inhibitor led to a significant repression of viral proliferation, implying that it acts as a positive regulator of PHEV proliferation. Using a dual-luciferase reporter assay, miR-142a-3p was found to bind directly bound to the 3' untranslated region (3'UTR) of Rab3a mRNA and downregulate its expression. Knockdown of Rab3a expression by transfection with an miR-142a-3p mimic or Rab3a siRNA significantly increased PHEV replication in N2a cells. Conversely, the use of an miR-142a-3p inhibitor or overexpression of Rab3a resulted in a marked restriction of viral production at both the mRNA and protein level. Our data demonstrate that miR-142a-3p promotes PHEV proliferation by directly targeting Rab3a mRNA, and this provides new insights into the mechanisms of PHEV-related pathogenesis and virus-host interactions.

The First Detection of Equine Coronavirus in Adult Horses and Foals in Ireland.

The objective of this study was to investigate the presence of equine coronavirus (ECoV) in clinical samples submitted to a diagnostic laboratory in Ireland. A total of 424 clinical samples were examined from equids with enteric disease in 24 Irish counties between 2011 and 2015. A real-time reverse transcription polymerase chain reaction was used to detect ECoV RNA. Nucleocapsid, spike and the region from the p4.7 to p12.7 genes of positive samples were sequenced, and sequence and phylogenetic analyses were conducted. Five samples (1.2%) collected in 2011 and 2013 tested positive for ECoV. Positive samples were collected from adult horses, Thoroughbred foals and a donkey foal. Sequence and/or phylogenetic analysis showed that nucleocapsid, spike and p12.7 genes were highly conserved and were closely related to ECoVs identified in other countries. In contrast, the region from p4.7 and the non-coding region following the p4.7 gene had deletions or insertions. The differences in the p4.7 region between the Irish ECoVs and other ECoVs indicated that the Irish viruses were distinguishable from those circulating in other countries. This is the first report of ECoV detected in both foals and adult horses in Ireland.

ATN-161 reduces virus proliferation in PHEV-infected mice by inhibiting the integrin α5ß1-FAK signaling pathway.

Porcine hemagglutinating encephalomyelitis virus (PHEV) is a typical neurotropic virus that can cause obvious nerve damage. Integrin α5ß1 is a transmembrane macromolecular that closely related to neurological function. We recently demonstrated that integrin α5ß1 plays a critical role in PHEV invasion in vitro. To determine the function and mechanism of integrin α5ß1 in virus proliferation in vivo, we established a mouse model of PHEV infection. Integrin α5ß1-FAK signaling pathway was activated in PHEV-infected mice by qPCR, Western blotting, and GST pull-down assays. Viral proliferation and integrin α5ß1-FAK signaling pathway were significantly inhibited after intravenous injection of ATN-161, an integrin α5ß1 inhibitor. Through a histological analysis, we found that ATN-161-treated mice only showed pathological changes in neuronal cytoplasmic swelling at 5 day post-infection. In summary, our results provide the first evidence that ATN-161 inhibits the proliferation of PHEV in mice and explores its underlying mechanisms of action.

Development of a triplex real-time RT-PCR assay for detection and differentiation of three US genotypes of porcine hemagglutinating encephalomyelitis virus.

Porcine hemagglutinating encephalomyelitis virus (PHEV) is a single-stranded, positive-sense RNA virus. PHEV mainly causes two types of clinical manifestations representing vomiting and wasting and encephalomyelitis in piglets. However, our recent findings provide strong evidence that PHEV can also cause respiratory disease in older pigs. Genomic analysis of new PHEV strains identified in our former study further classifies PHEV into three genotypes. Detection and differentiation of these new mutants are critical in monitoring PHEV evolution in the field. In the present study, we report the development of a triplex real-time RT-PCR assay for detection and differentiation of three PHEV genotypes, 1, 2, and 3. Three sets of primers and probes were designed; one set of primers and probe targeting the conserved regions of the 3' end nucleocapsid for detection of all three genotypes and another two sets of primers and probes targeting the regions of NS2 with different patterns of deletions for detection of both genotypes 1 and 3, or genotype 3 only. Genotype 1 was positive when two probe dyes showed signals, genotype 2 was positive when only one probe dye showed a signal, and genotype 3 was positive when all three probes showed signals. The detection limit of the developed triplex real-time RT-PCR was as low as 8 or 9 DNA copies for three sets of primers and probes. The specificity test showed no cross reaction with other porcine viruses. Positive field-samples were correctly typed by this new assay, which was further confirmed by DNA sequencing. The triplex real-time RT-PCR provides a rapid and sensitive method to detect and differentiate all three US genotypes of PHEV from clinical samples.

Genomic characterization and pathogenicity of a porcine hemagglutinating encephalomyelitis virus strain isolated in China.

Porcine hemagglutinating encephalomyelitis virus (PHEV) is a member of the genus betacoronavirus within the family coronaviridae, which invades the central nervous system (CNS) via peripheral nervous system and causes encephalomyelitis or vomiting and wasting disease (VWD) in sucking piglets. Up to now, although few complete nucleotide sequences of PHEV have been reported, they are not annotated. This study aimed to illuminate genome characterization, phylogenesis and pathogenicity of the PHEV/2008 strain. The full length of the PHEV/2008 strain genome was 30,684 bp, with a G + C content of 37.27%. The genome included at a minimum of 11 predicted open reading frames (ORFs) flanked by 5' and 3' untranslated regions (UTR) of 211 and 289 nucleotides. The replicase polyproteins pp1a and pp1ab, which had 4382 and 7094 amino acid residues, respectively, were predicted to be cleaved into 16 subunits by two viral proteinases. Phylogenetic analysis based on the complete genome sequence revealed that PHEV/2008 strain was genetically different from other known PHEV types, which represented a novel genotype (GI-1). In addition, we found that PHEV/2008 was neurotropic and highly pathogenic to 4-week-old BALB/c mice. Taken together, this is the first detailed annotated, complete genomic sequence of a new genotype PHEV strain in China.

Porcine Hemagglutinating Encephalomyelitis Virus Enters Neuro-2a Cells via Clathrin-Mediated Endocytosis in a Rab5-, Cholesterol-, and pH-Dependent Manner.

Porcine hemagglutinating encephalomyelitis virus (PHEV) is a highly neurovirulent coronavirus that invades the central nervous system (CNS) in piglets. Although important progress has been made toward understanding the biology of PHEV, many aspects of its life cycle remain obscure. Here we dissected the molecular mechanism underlying cellular entry and intracellular trafficking of PHEV in mouse neuroblastoma (Neuro-2a) cells. We first performed a thin-section transmission electron microscopy (TEM) assay to characterize the kinetics of PHEV, and we found that viral entry and transfer occur via membranous coating-mediated endo- and exocytosis. To verify the roles of distinct endocytic pathways, systematic approaches were used, including pharmacological inhibition, RNA interference, confocal microscopy analysis, use of fluorescently labeled virus particles, and overexpression of a dominant negative (DN) mutant. Quantification of infected cells showed that PHEV enters cells by clathrin-mediated endocytosis (CME) and that low pH, dynamin, cholesterol, and Eps15 are indispensably involved in this process. Intriguingly, PHEV invasion leads to rapid actin rearrangement, suggesting that the intactness and dynamics of the actin cytoskeleton are positively correlated with viral endocytosis. We next investigated the trafficking of internalized PHEV and found that Rab5- and Rab7-dependent pathways are required for the initiation of a productive infection. Furthermore, a GTPase activation assay suggested that endogenous Rab5 is activated by PHEV and is crucial for viral progression. Our findings demonstrate that PHEV hijacks the CME and endosomal system of the host to enter and traffic within neural cells, providing new insights into PHEV pathogenesis and guidance for antiviral drug design.IMPORTANCE Porcine hemagglutinating encephalomyelitis virus (PHEV), a nonsegmented, positive-sense, single-stranded RNA coronavirus, invades the central nervous system (CNS) and causes neurological dysfunction. Neural cells are its targets for viral progression. However, the detailed mechanism underlying PHEV entry and trafficking remains unknown. PHEV is the etiological agent of porcine hemagglutinating encephalomyelitis, which is an acute and highly contagious disease that causes numerous deaths in suckling piglets and enormous economic losses in China. Understanding the viral entry pathway will not only advance our knowledge of PHEV infection and pathogenesis but also open new approaches to the development of novel therapeutic strategies. Therefore, we employed systematic approaches to dissect the internalization and intracellular trafficking mechanism of PHEV in Neuro-2a cells. This is the first report to describe the process of PHEV entry into nerve cells via clathrin-mediated endocytosis in a dynamin-, cholesterol-, and pH-dependent manner that requires Rab5 and Rab7.

Porcine Hemagglutinating Encephalomyelitis Virus and Respiratory Disease in Exhibition Swine, Michigan, USA, 2015.

Acute outbreaks of respiratory disease in swine at agricultural fairs in Michigan, USA, in 2015 raised concern for potential human exposure to influenza A virus. Testing ruled out influenza A virus and identified porcine hemagglutinating encephalomyelitis virus as the cause of influenza-like illness in the affected swine.

Discovery of a novel canine respiratory coronavirus support genetic recombination among betacoronavirus1.

Although canine respiratory coronavirus (CRCoV) is an important respiratory pathogen that is prevalent in many countries, only one complete genome sequence of CRCoV (South Korea strain K37) has been obtained to date. Genome-wide analyses and recombination have rarely been conducted, as small numbers of samples and limited genomic characterization have previously prevented further analyses. Herein, we report a unique CRCoV strain, denoted strain BJ232, derived from a CRCoV-positive dog with a mild respiratory infection. Phylogenetic analysis based on complete genome of all available coronaviruses consistently show that CRCoV BJ232 is most closely related to human coronavirus OC43 (HCoV-OC43) and BCoV, forming a separate clade that split off early from other Betacoronavirus 1. Based on the phylogenetic and SimPlot analysis we propose that CRCoV-K37 was derived from genetic recombination between CRCoV-BJ232 and BCoV. In detail, spike (S) gene of CRCoV-K37 clustered with CRCoV-BJ232. However orf1ab, membrane (M) and nucleocapsid (N) genes were more related to Bovine coronavirus (BCoV) than CRCoV-B232. Molecular epidemic analysis confirmed the prevalence of CRCoV-BJ232 lineage around the world for a long time. Recombinant events among Betacoronavirus 1 may have implications for CRCoV transmissibility. All these findings provide further information regarding the origin of CRCoV.