Publications by authors named "Mia Madel Alfajaro"

47 Publications

Discovery and functional interrogation of SARS-CoV-2 RNA-host protein interactions.

Cell 2021 Mar 11. Epub 2021 Mar 11.

Department of Pathology, Stanford University, Stanford, CA, USA. Electronic address:

SARS-CoV-2 is the cause of a pandemic with growing global mortality. Using comprehensive identification of RNA-binding proteins by mass spectrometry (ChIRP-MS), we identified 309 host proteins that bind the SARS-CoV-2 RNA during active infection. Integration of this data with ChIRP-MS data from three other RNA viruses defined viral specificity of RNA-host protein interactions. Targeted CRISPR screens revealed that the majority of functional RNA-binding proteins protect the host from virus-induced cell death, and comparative CRISPR screens across seven RNA viruses revealed shared and SARS-specific antiviral factors. Finally, by combining the RNA-centric approach and functional CRISPR screens, we demonstrated a physical and functional connection between SARS-CoV-2 and mitochondria, highlighting this organelle as a general platform for antiviral activity. Altogether, these data provide a comprehensive catalog of functional SARS-CoV-2 RNA-host protein interactions, which may inform studies to understand the host-virus interface and nominate host pathways that could be targeted for therapeutic benefit.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.cell.2021.03.012DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7951565PMC
March 2021

Single-cell longitudinal analysis of SARS-CoV-2 infection in human airway epithelium identifies target cells, alterations in gene expression, and cell state changes.

PLoS Biol 2021 03 17;19(3):e3001143. Epub 2021 Mar 17.

Department of Laboratory Medicine, Yale University, New Haven, Connecticut, United States of America.

There are currently limited Food and Drug Administration (FDA)-approved drugs and vaccines for the treatment or prevention of Coronavirus Disease 2019 (COVID-19). Enhanced understanding of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection and pathogenesis is critical for the development of therapeutics. To provide insight into viral replication, cell tropism, and host-viral interactions of SARS-CoV-2, we performed single-cell (sc) RNA sequencing (RNA-seq) of experimentally infected human bronchial epithelial cells (HBECs) in air-liquid interface (ALI) cultures over a time course. This revealed novel polyadenylated viral transcripts and highlighted ciliated cells as a major target at the onset of infection, which we confirmed by electron and immunofluorescence microscopy. Over the course of infection, the cell tropism of SARS-CoV-2 expands to other epithelial cell types including basal and club cells. Infection induces cell-intrinsic expression of type I and type III interferons (IFNs) and interleukin (IL)-6 but not IL-1. This results in expression of interferon-stimulated genes (ISGs) in both infected and bystander cells. This provides a detailed characterization of genes, cell types, and cell state changes associated with SARS-CoV-2 infection in the human airway.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1371/journal.pbio.3001143DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8007021PMC
March 2021

Non-steroidal anti-inflammatory drugs dampen the cytokine and antibody response to SARS-CoV-2 infection.

J Virol 2021 Jan 13. Epub 2021 Jan 13.

Department of Laboratory Medicine, Yale University School of Medicine, New Haven, CT, USA

Identifying drugs that regulate severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and its symptoms has been a pressing area of investigation during the coronavirus disease 2019 (COVID-19) pandemic. Nonsteroidal anti-inflammatory drugs (NSAIDs), which are frequently used for the relief of pain and inflammation, could modulate both SARS-CoV-2 infection and the host response to the virus. NSAIDs inhibit the enzymes cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2), which mediate the production of prostaglandins (PGs). As PGs play diverse biological roles in homeostasis and inflammatory responses, inhibiting PG production with NSAIDs could affect COVID-19 pathogenesis in multiple ways, including: (1) altering susceptibility to infection by modifying expression of angiotensin-converting enzyme 2 (ACE2), the cell entry receptor for SARS-CoV-2; (2) regulating replication of SARS-CoV-2 in host cells; and (3) modulating the immune response to SARS-CoV-2. Here, we investigate these potential roles. We demonstrate that SARS-CoV-2 infection upregulates COX-2 in diverse human cell culture and mouse systems. However, suppression of COX-2 by two commonly used NSAIDs, ibuprofen and meloxicam, had no effect on expression, viral entry, or viral replication. In contrast, in a mouse model of SARS-CoV-2 infection, NSAID treatment reduced production of pro-inflammatory cytokines and impaired the humoral immune response to SARS-CoV-2 as demonstrated by reduced neutralizing antibody titers. Our findings indicate that NSAID treatment may influence COVID-19 outcomes by dampening the inflammatory response and production of protective antibodies rather than modifying susceptibility to infection or viral replication.Public health officials have raised concerns about the use of nonsteroidal anti-inflammatory drugs (NSAIDs) for treating symptoms of coronavirus disease 2019 (COVID-19). NSAIDs inhibit the enzymes cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2), which are critical for the generation of prostaglandins - lipid molecules with diverse roles in homeostasis and inflammation. Inhibition of prostaglandin production by NSAIDs could therefore have multiple effects on COVID-19 pathogenesis. Here, we demonstrate that NSAID treatment reduced both the antibody and pro-inflammatory cytokine response to SARS-CoV-2 infection. The ability of NSAIDs to modulate the immune response to SARS-CoV-2 infection has important implications for COVID-19 pathogenesis in patients. Whether this occurs in humans and whether it is beneficial or detrimental to the host remains an important area of future investigation. This also raises the possibility that NSAIDs may alter the immune response to SARS-CoV-2 vaccination.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1128/JVI.00014-21DOI Listing
January 2021

Neuroinvasion of SARS-CoV-2 in human and mouse brain.

J Exp Med 2021 03;218(3)

Department of Immunobiology, Yale School of Medicine, New Haven, CT.

Although COVID-19 is considered to be primarily a respiratory disease, SARS-CoV-2 affects multiple organ systems including the central nervous system (CNS). Yet, there is no consensus on the consequences of CNS infections. Here, we used three independent approaches to probe the capacity of SARS-CoV-2 to infect the brain. First, using human brain organoids, we observed clear evidence of infection with accompanying metabolic changes in infected and neighboring neurons. However, no evidence for type I interferon responses was detected. We demonstrate that neuronal infection can be prevented by blocking ACE2 with antibodies or by administering cerebrospinal fluid from a COVID-19 patient. Second, using mice overexpressing human ACE2, we demonstrate SARS-CoV-2 neuroinvasion in vivo. Finally, in autopsies from patients who died of COVID-19, we detect SARS-CoV-2 in cortical neurons and note pathological features associated with infection with minimal immune cell infiltrates. These results provide evidence for the neuroinvasive capacity of SARS-CoV-2 and an unexpected consequence of direct infection of neurons by SARS-CoV-2.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1084/jem.20202135DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7808299PMC
March 2021

CD300lf Conditional Knockout Mouse Reveals Strain-Specific Cellular Tropism of Murine Norovirus.

J Virol 2021 01 13;95(3). Epub 2021 Jan 13.

Department of Laboratory Medicine, Yale University School of Medicine, New Haven, Connecticut, USA

Noroviruses are a leading cause of gastrointestinal infection in humans and mice. Understanding human norovirus (HuNoV) cell tropism has important implications for our understanding of viral pathogenesis. Murine norovirus (MNoV) is extensively used as a surrogate model for HuNoV. We previously identified CD300lf as the receptor for MNoV. Here, we generated a conditional knockout ( ) mouse to elucidate the cell tropism of persistent and nonpersistent strains of murine norovirus. Using this mouse model, we demonstrated that CD300lf expression on intestinal epithelial cells (IECs), and on tuft cells in particular, is essential for transmission of the persistent MNoV strain CR6 (MNoV) In contrast, the nonpersistent MNoV strain CW3 (MNoV) does not require CD300lf expression on IECs for infection. However, deletion of CD300lf in myelomonocytic cells (+) partially reduces CW3 viral load in lymphoid and intestinal tissues. Disruption of CD300lf expression on B cells (), neutrophils (), and dendritic cells () did not affect MNoV viral RNA levels. Finally, we show that the transcription factor STAT1, which is critical for the innate immune response, partially restricts the cell tropism of MNoV to LysM+ cells. Taken together, these data demonstrate that CD300lf expression on tuft cells is essential for MNoV; that myelomonocytic cells are a major, but not exclusive, target cell of MNoV; and that STAT1 signaling restricts the cellular tropism of MNoV This study provides the first genetic system for studying the cell type-specific role of CD300lf in norovirus pathogenesis. Human noroviruses (HuNoVs) are a leading cause of gastroenteritis resulting in up to 200,000 deaths each year. The receptor and cell tropism of HuNoV in immunocompetent humans are unclear. We use murine norovirus (MNoV) as a model for HuNoV. We recently identified CD300lf as the sole physiologic receptor for MNoV. Here, we leverage this finding to generate a conditional knockout mouse to decipher the contributions of specific cell types to MNoV infection. We demonstrate that persistent MNoV requires CD300lf expression on tuft cells. In contrast, multiple CD300lf+ cell types, dominated by myelomonocytic cells, are sufficient for nonpersistent MNoV infection. CD300lf expression on epithelial cells, B cells, neutrophils, and dendritic cells is not critical for MNoV infection. Mortality associated with the MNoV strain in mice does not require CD300lf expression on LysM+ cells, highlighting that both CD300lf receptor expression and innate immunity regulate MNoV cell tropism .
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1128/JVI.01652-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7925115PMC
January 2021

Genome-wide CRISPR Screens Reveal Host Factors Critical for SARS-CoV-2 Infection.

Cell 2021 01 20;184(1):76-91.e13. Epub 2020 Oct 20.

Department of Laboratory Medicine, Yale School of Medicine, New Haven, CT 06520, USA; Department of Immunobiology, Yale School of Medicine, New Haven, CT 06520, USA; Yale Cancer Center, Yale School of Medicine, New Haven, CT 06520, USA. Electronic address:

Identification of host genes essential for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection may reveal novel therapeutic targets and inform our understanding of coronavirus disease 2019 (COVID-19) pathogenesis. Here we performed genome-wide CRISPR screens in Vero-E6 cells with SARS-CoV-2, Middle East respiratory syndrome CoV (MERS-CoV), bat CoV HKU5 expressing the SARS-CoV-1 spike, and vesicular stomatitis virus (VSV) expressing the SARS-CoV-2 spike. We identified known SARS-CoV-2 host factors, including the receptor ACE2 and protease Cathepsin L. We additionally discovered pro-viral genes and pathways, including HMGB1 and the SWI/SNF chromatin remodeling complex, that are SARS lineage and pan-coronavirus specific, respectively. We show that HMGB1 regulates ACE2 expression and is critical for entry of SARS-CoV-2, SARS-CoV-1, and NL63. We also show that small-molecule antagonists of identified gene products inhibited SARS-CoV-2 infection in monkey and human cells, demonstrating the conserved role of these genetic hits across species. This identifies potential therapeutic targets for SARS-CoV-2 and reveals SARS lineage-specific and pan-CoV host factors that regulate susceptibility to highly pathogenic CoVs.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.cell.2020.10.028DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7574718PMC
January 2021

Systematic discovery and functional interrogation of SARS-CoV-2 viral RNA-host protein interactions during infection.

bioRxiv 2020 Oct 6. Epub 2020 Oct 6.

Department of Pathology, Stanford University, Stanford, CA.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the cause of a pandemic with growing global mortality. There is an urgent need to understand the molecular pathways required for host infection and anti-viral immunity. Using comprehensive identification of RNA-binding proteins by mass spectrometry (ChIRP-MS), we identified 309 host proteins that bind the SARS-CoV-2 RNA during active infection. Integration of this data with viral ChIRP-MS data from three other positive-sense RNA viruses defined pan-viral and SARS-CoV-2-specific host interactions. Functional interrogation of these factors with a genome-wide CRISPR screen revealed that the vast majority of viral RNA-binding proteins protect the host from virus-induced cell death, and we identified known and novel anti-viral proteins that regulate SARS-CoV-2 pathogenicity. Finally, our RNA-centric approach demonstrated a physical connection between SARS-CoV-2 RNA and host mitochondria, which we validated with functional and electron microscopy data, providing new insights into a more general virus-specific protein logic for mitochondrial interactions. Altogether, these data provide a comprehensive catalogue of SARS-CoV-2 RNA-host protein interactions, which may inform future studies to understand the mechanisms of viral pathogenesis, as well as nominate host pathways that could be targeted for therapeutic benefit.

Highlights: · ChIRP-MS of SARS-CoV-2 RNA identifies a comprehensive viral RNA-host protein interaction network during infection across two species· Comparison to RNA-protein interaction networks with Zika virus, dengue virus, and rhinovirus identify SARS-CoV-2-specific and pan-viral RNA protein complexes and highlights distinct intracellular trafficking pathways· Intersection of ChIRP-MS and genome-wide CRISPR screens identify novel SARS-CoV-2-binding proteins with pro- and anti-viral function· Viral RNA-RNA and RNA-protein interactions reveal specific SARS-CoV-2-mediated mitochondrial dysfunction during infection.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1101/2020.10.06.327445DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7553159PMC
October 2020

Cyclooxgenase-2 is induced by SARS-CoV-2 infection but does not affect viral entry or replication.

bioRxiv 2020 Sep 25. Epub 2020 Sep 25.

Identifying drugs that regulate severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and its symptoms has been a pressing area of investigation during the coronavirus disease 2019 (COVID-19) pandemic. Nonsteroidal anti-inflammatory drugs (NSAIDs), which are frequently used for the relief of pain and inflammation, could modulate both SARS-CoV-2 infection and the host response to the virus. NSAIDs inhibit the enzymes cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2), which mediate the production of prostaglandins (PGs). PGE , one of the most abundant PGs, has diverse biological roles in homeostasis and inflammatory responses. Previous studies have shown that NSAID treatment or inhibition of PGE receptor signaling leads to upregulation of angiotensin-converting enzyme 2 (ACE2), the cell entry receptor for SARS-CoV-2, thus raising concerns that NSAIDs could increase susceptibility to infection. COX/PGE signaling has also been shown to regulate the replication of many viruses, but it is not yet known whether it plays a role in SARS-CoV-2 replication. The purpose of this study was to dissect the effect of NSAIDs on COVID-19 in terms of SARS-CoV-2 entry and replication. We found that SARS-CoV-2 infection induced COX-2 upregulation in diverse human cell culture and mouse systems. However, suppression of COX-2/PGE signaling by two commonly used NSAIDs, ibuprofen and meloxicam, had no effect on expression, viral entry, or viral replication. Our findings suggest that COX-2 signaling driven by SARS-CoV-2 may instead play a role in regulating the lung inflammation and injury observed in COVID-19 patients.

Importance: Public health officials have raised concerns about the use of nonsteroidal anti-inflammatory drugs (NSAIDs) for treating symptoms of coronavirus disease 2019 (COVID-19), which is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). NSAIDs function by inhibiting the enzymes cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2). These enzymes are critical for the generation of prostaglandins, lipid molecules with diverse roles in maintaining homeostasis as well as regulating the inflammatory response. While COX-1/COX-2 signaling pathways have been shown to affect the replication of many viruses, their effect on SARS-CoV-2 infection remains unknown. We found that SARS-CoV-2 infection induced COX-2 expression in both human cell culture systems and mouse models. However, inhibition of COX-2 activity with NSAIDs did not affect SARS-CoV-2 entry or replication. Our findings suggest that COX-2 signaling may instead regulate the lung inflammation observed in COVID-19 patients, which is an important area for future studies.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1101/2020.09.24.312769DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7523115PMC
September 2020

Neuroinvasion of SARS-CoV-2 in human and mouse brain.

bioRxiv 2020 Sep 8. Epub 2020 Sep 8.

Department of Immunobiology, Yale School of Medicine, New Haven, CT 06510, USA.

Although COVID-19 is considered to be primarily a respiratory disease, SARS-CoV-2 affects multiple organ systems including the central nervous system (CNS). Yet, there is no consensus whether the virus can infect the brain, or what the consequences of CNS infection are. Here, we used three independent approaches to probe the capacity of SARS-CoV-2 to infect the brain. First, using human brain organoids, we observed clear evidence of infection with accompanying metabolic changes in the infected and neighboring neurons. However, no evidence for the type I interferon responses was detected. We demonstrate that neuronal infection can be prevented either by blocking ACE2 with antibodies or by administering cerebrospinal fluid from a COVID-19 patient. Second, using mice overexpressing human ACE2, we demonstrate that SARS-CoV-2 neuroinvasion, but not respiratory infection, is associated with mortality. Finally, in brain autopsy from patients who died of COVID-19, we detect SARS-CoV-2 in the cortical neurons, and note pathologic features associated with infection with minimal immune cell infiltrates. These results provide evidence for the neuroinvasive capacity of SARS-CoV2, and an unexpected consequence of direct infection of neurons by SARS-CoV-2.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1101/2020.06.25.169946DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7491522PMC
September 2020

Genome-wide CRISPR screen reveals host genes that regulate SARS-CoV-2 infection.

bioRxiv 2020 Jun 17. Epub 2020 Jun 17.

Identification of host genes essential for SARS-CoV-2 infection may reveal novel therapeutic targets and inform our understanding of COVID-19 pathogenesis. Here we performed a genome-wide CRISPR screen with SARS-CoV-2 and identified known SARS-CoV-2 host factors including the receptor ACE2 and protease Cathepsin L. We additionally discovered novel pro-viral genes and pathways including the SWI/SNF chromatin remodeling complex and key components of the TGF-β signaling pathway. Small molecule inhibitors of these pathways prevented SARS-CoV-2-induced cell death. We also revealed that the alarmin HMGB1 is critical for SARS-CoV-2 replication. In contrast, loss of the histone H3.3 chaperone complex sensitized cells to virus-induced death. Together this study reveals potential therapeutic targets for SARS-CoV-2 and highlights host genes that may regulate COVID-19 pathogenesis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1101/2020.06.16.155101DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7457610PMC
June 2020

Mouse model of SARS-CoV-2 reveals inflammatory role of type I interferon signaling.

J Exp Med 2020 12;217(12)

Department of Immunobiology, Yale University School of Medicine, New Haven, CT.

Severe acute respiratory syndrome-coronavirus 2 (SARS-Cov-2) has caused over 13,000,000 cases of coronavirus disease (COVID-19) with a significant fatality rate. Laboratory mice have been the stalwart of therapeutic and vaccine development; however, they do not support infection by SARS-CoV-2 due to the virus's inability to use the mouse orthologue of its human entry receptor angiotensin-converting enzyme 2 (hACE2). While hACE2 transgenic mice support infection and pathogenesis, these mice are currently limited in availability and are restricted to a single genetic background. Here we report the development of a mouse model of SARS-CoV-2 based on adeno-associated virus (AAV)-mediated expression of hACE2. These mice support viral replication and exhibit pathological findings found in COVID-19 patients. Moreover, we show that type I interferons do not control SARS-CoV-2 replication in vivo but are significant drivers of pathological responses. Thus, the AAV-hACE2 mouse model enables rapid deployment for in-depth analysis following robust SARS-CoV-2 infection with authentic patient-derived virus in mice of diverse genetic backgrounds.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1084/jem.20201241DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7401025PMC
December 2020

Mouse Model of SARS-CoV-2 Reveals Inflammatory Role of Type I Interferon Signaling.

SSRN 2020 Jun 16:3628297. Epub 2020 Jun 16.

Department of Immunobiology, Yale University School of Medicine, New Haven, CT, USA.

Severe Acute Respiratory Syndrome- Coronavirus 2 (SARS-Cov-2) has caused over 5,000,000 cases of Coronavirus disease (COVID-19) with significant fatality rate. Due to the urgency of this global pandemic, numerous therapeutic and vaccine trials have begun without customary safety and efficacy studies.  Laboratory mice have been the stalwart of these types of studies; however, they do not support infection by SARS-CoV-2 due to the inability of its spike (S) protein to engage the mouse ortholog of its human entry receptor angiotensin-converting enzyme 2 (hACE2). While hACE2 transgenic mice support infection and pathogenesis, these mice are currently limited in availability and are restricted to a single genetic background. Here we report the development of a mouse model of SARS-CoV-2 based on adeno associated virus (AAV)-mediated expression of hACE2. These mice support viral replication and antibody production and exhibit pathologic findings found in COVID-19 patients as well as non-human primate models. Moreover, we show that type I interferons are unable to control SARS-CoV2 replication and drive pathologic responses. Thus, the hACE2-AAV mouse model enables rapid deployment for in-depth analysis following robust SARS-CoV-2 infection with authentic patient-derived virus in mice of diverse genetic backgrounds.  This represents a much-needed platform for rapidly testing prophylactic and therapeutic strategies to combat COVID-19. Funding: This study was supported by awards from National Institute of Health grants, 2T32AI007517-16 (to BI), T32GM007205 and F30CA239444 (to ES), AI054359 and AI127429 (to AI), T32AI007019 (to TM),K08 AI128043 (to CBW), as well as Women's Health Research at Yale Pilot Project Program (AI, AR), Fast Grant from Emergent Ventures at the Mercatus Center (AI, ES), Mathers Foundation (AR, CBW, AI), and the Ludwig Family Foundation (AI, AR, CBW). A.I. is an investigator of the Howard Hughes Medical Institute. Conflict of Interest: None of the authors declare interests related to the manuscript. Ethical Approval: All procedures were performed in a BSL-3 facility (for SARS-CoV-2 infected mice) with approval from the Yale Environmental Health and Safety office.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.2139/ssrn.3628297DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7366812PMC
June 2020

Perfusion change in benign prostatic hyperplasia before and after castration in a canine model: Contrast enhanced ultrasonography and CT perfusion study.

Theriogenology 2020 Oct 1;156:97-106. Epub 2020 Jul 1.

Veterinary Medical Imaging, College of Veterinary Medicine, Chonnam National University, Gwangju, 61186, Republic of Korea. Electronic address:

Hormonal and vascular changes affecting the canine prostate after castration were investigated to identify the effects of hormones and perfusion on the development of benign prostatic hyperplasia (BPH). Concentrations of serum testosterone and estrogen and intraprostatic dihydrotestosterone (DHT) were compared between 5 normal dogs, 6 dogs with mild BPH, and 6 dogs with marked BPH. In addition, prostatic perfusion using contrast-enhanced ultrasonography (CEUS) and CT perfusion (CTP), as well as CT volumes of the prostates were compared. The changes in these values following castration were assessed in 6 dogs with marked BPH. CEUS revealed significantly slower prostatic arterial inflow and relatively faster venous outflow in BPH versus normal prostate. Permeability and blood volume were not significantly different between the groups via CTP. Intraprostatic DHT level was higher in BPH than in normal prostate and decreased significantly following castration, which was accompanied by a rapid decrease in prostatic volume. On CEUS, arterial inflow to the prostate significantly decreased following castration. Blood volume within the prostate decreased significantly by day 60 following castration. Permeability increased significantly during the early phase after castration; however, by day 60 post-castration, all perfusion parameters decreased significantly. Perfusion changes including venous parameters measured by CEUS and blood volume changes measured by CTP, however, did not support the backflow theory postulating that BPH is induced by vascular changes from congested testes. The major etiology for the development of BPH is attributed to be increased levels of DHT rather than vascular changes.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.theriogenology.2020.06.026DOI Listing
October 2020

Mouse model of SARS-CoV-2 reveals inflammatory role of type I interferon signaling.

bioRxiv 2020 May 27. Epub 2020 May 27.

Department of Immunobiology, Yale University School of Medicine, New Haven, CT, USA.

Severe Acute Respiratory Syndrome- Coronavirus 2 (SARS-Cov-2) has caused over 5,000,000 cases of Coronavirus disease (COVID-19) with significant fatality rate. Due to the urgency of this global pandemic, numerous therapeutic and vaccine trials have begun without customary safety and efficacy studies. Laboratory mice have been the stalwart of these types of studies; however, they do not support infection by SARS-CoV-2 due to the inability of its spike (S) protein to engage the mouse ortholog of its human entry receptor angiotensin-converting enzyme 2 (hACE2). While hACE2 transgenic mice support infection and pathogenesis, these mice are currently limited in availability and are restricted to a single genetic background. Here we report the development of a mouse model of SARS-CoV-2 based on adeno associated virus (AAV)-mediated expression of hACE2. These mice support viral replication and antibody production and exhibit pathologic findings found in COVID-19 patients as well as non-human primate models. Moreover, we show that type I interferons are unable to control SARS-CoV2 replication and drive pathologic responses. Thus, the hACE2-AAV mouse model enables rapid deployment for in-depth analysis following robust SARS-CoV-2 infection with authentic patient-derived virus in mice of diverse genetic backgrounds. This represents a much-needed platform for rapidly testing prophylactic and therapeutic strategies to combat COVID-19.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1101/2020.05.27.118893DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7302201PMC
May 2020

Single-cell longitudinal analysis of SARS-CoV-2 infection in human bronchial epithelial cells.

bioRxiv 2020 May 7. Epub 2020 May 7.

Department of Laboratory Medicine, Yale School of Medicine, New Haven, CT, USA.

SARS-CoV-2, the causative agent of COVID-19, has resulted in more than 3,000,000 infections and 200,000 deaths. There are currently no approved drugs or vaccines for the treatment or prevention of COVID-19. Enhanced understanding of SARS-CoV-2 infection and pathogenesis is critical for the development of therapeutics. To reveal insight into viral replication, cell tropism, and host-viral interactions of SARS-CoV-2 we performed single-cell RNA sequencing of experimentally infected human bronchial epithelial cells (HBECs) in air-liquid interface cultures over a time-course. This revealed novel polyadenylated viral transcripts and highlighted ciliated cells as the major target of infection, which we confirmed by electron microscopy. Over the course of infection, cell tropism of SARS-CoV-2 expands to other epithelial cell types including basal and club cells. Infection induces cell intrinsic expression of type I and type III IFNs and IL6 but not IL1. This results in expression of interferon stimulated genes in both infected and bystander cells. Here, we have conducted an in-depth analysis of SARS-CoV-2 infection in HBECs and provide a detailed characterization of genes, cell types, and cell state changes associated with the infection.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1101/2020.05.06.081695DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7263511PMC
May 2020

Dual Recognition of Sialic Acid and αGal Epitopes by the VP8* Domains of the Bovine Rotavirus G6P[5] WC3 and of Its Mono-reassortant G4P[5] RotaTeq Vaccine Strains.

J Virol 2019 09 28;93(18). Epub 2019 Aug 28.

Laboratory of Veterinary Pathology, College of Veterinary Medicine, Chonnam National University, Gwangju, Republic of Korea

Group A rotaviruses, an important cause of severe diarrhea in children and young animals, initiate infection via interactions of the VP8* domain of the VP4 spike protein with cell surface sialic acids (SAs) or histo-blood group antigens (HBGAs). Although the bovine G6P[5] WC3 strain is an important animal pathogen and is also used in the bovine-human reassortant RotaTeq vaccine, the receptor(s) for the VP8* domain of WC3 and its reassortant strains have not yet been identified. In the present study, HBGA- and saliva-binding assays showed that both G6P[5] WC3 and mono-reassortant G4P[5] strains recognized the αGal HBGA. The infectivity of both P[5]-bearing strains was significantly reduced in αGal-free MA-104 cells by pretreatment with a broadly specific neuraminidase or by coincubation with the α2,6-linked SA-specific lectin, but not by the α2,3-linked specific sialidase or by lectin. Free NeuAc and the αGal trisaccharide also prevented the infectivity of both strains. This indicated that both P[5]-bearing strains utilize α2,6-linked SA as a ligand on MA104 cells. However, the two strains replicated in differentiated bovine small intestinal enteroids and in their human counterparts that lack α2,6-linked SA or αGal HBGA, suggesting that additional or alternative receptors such as integrins, hsp70, and tight-junction proteins bound directly to the VP5* domain can be used by the P[5]-bearing strains to initiate the infection of human cells. In addition, these data also suggested that P[5]-bearing strains have potential for cross-species transmission. Group A rotaviruses initiate infection through the binding of the VP8* domain of the VP4 protein to sialic acids (SAs) or histo-blood group antigens (HBGAs). Although the bovine G6P[5] WC3 strain is an important animal pathogen and is used as the backbone in the bovine-human reassortant RotaTeq vaccine, the receptor(s) for their P[5] VP8* domain has remained elusive. Using a variety of approaches, we demonstrated that the WC3 and bovine-human mono-reassortant G4P[5] vaccine strains recognize both α2,6-linked SA and αGal HBGA as ligands. Neither ligand is expressed on human small intestinal epithelial cells, explaining the absence of natural human infection by P[5]-bearing strains. However, we observed that the P[5]-bearing WC3 and G4P[5] RotaTeq vaccine strains could still infect human intestinal epithelial cells. Thus, the four P[5] RotaTeq vaccine strains potentially binding to additional alternative receptors may be efficient and effective in providing protection against severe rotavirus disease in human.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1128/JVI.00941-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6714814PMC
September 2019

Development of a live attenuated trivalent porcine rotavirus A vaccine against disease caused by recent strains most prevalent in South Korea.

Vet Res 2019 Jan 7;50(1). Epub 2019 Jan 7.

Laboratory of Veterinary Pathology, College of Veterinary Medicine, Chonnam National University, Gwangju, Republic of Korea.

Porcine rotaviruses cause severe economic losses in the Korean swine industry due to G- and P-genotype mismatches between the predominant field and vaccine strains. Here, we developed a live attenuated trivalent porcine group A rotavirus vaccine using 80 cell culture passages of the representative Korean predominant strains G8P[7] 174-1, G9P[23] PRG942, and G5P[7] K71. Vaccination with the trivalent vaccine or its individual components induced no diarrhea during the first 2 weeks post-vaccination, i.e., the vaccines were attenuated. Challenge of trivalent-vaccinated or component-vaccinated piglets with homologous virulent strain(s) did not induce diarrhea for 2 weeks post-challenge. Immunization with the trivalent vaccine or its individual components also alleviated the histopathological lesions in the small intestines caused by challenge with the corresponding original virulent strain(s). Fecal secretory IgAs specific for each of vaccine strains were detected starting at 14 days post-vaccination (dpv), and IgA levels gradually increased up to 28 dpv. Oral immunization with the trivalent vaccine or its individual components induced high levels of serum virus-neutralizing antibody by 7 dpv. No diarrhea was observed in any experimental piglets during five consecutive passages of each vaccine strain. Our data indicated that the live attenuated trivalent vaccine was safe and effective at protecting piglets from diarrhea induced by challenge exposure of homologous virulent strains. This trivalent vaccine will potentially contribute toward controlling porcine rotavirus disease in South Korea and other countries where rotavirus infections with similar G and P genotypes are problematic.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/s13567-018-0619-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6323864PMC
January 2019

Early Porcine Sapovirus Infection Disrupts Tight Junctions and Uses Occludin as a Coreceptor.

J Virol 2019 02 5;93(4). Epub 2019 Feb 5.

Laboratory of Veterinary Pathology, College of Veterinary Medicine, Chonnam National University, Gwangju, Republic of Korea

The genus belongs to the family , and its members are common causative agents of severe acute gastroenteritis in both humans and animals. Some caliciviruses are known to use either terminal sialic acids or histo-blood group antigens as attachment factors and/or cell surface proteins, such as CD300lf, CD300ld, and junctional adhesion molecule 1 of tight junctions (TJs), as receptors. However, the roles of TJs and their proteins in sapovirus entry have not been examined. In this study, we found that porcine sapovirus (PSaV) significantly decreased transepithelial electrical resistance and increased paracellular permeability early in infection of LLC-PK cells, suggesting that PSaV dissociates TJs of cells. This led to the interaction between PSaV particles and occludin, which traveled in a complex into late endosomes via Rab5- and Rab7-dependent trafficking. Inhibition of occludin using small interfering RNA (siRNA), a specific antibody, or a dominant-negative mutant significantly blocked the entry of PSaV. Transient expression of occludin in nonpermissive Chinese hamster ovary (CHO) cells conferred susceptibility to PSaV, but only for a limited time. Although claudin-1, another TJ protein, neither directly interacted nor was internalized with PSaV particles, it facilitated PSaV entry and replication in the LLC-PK cells. We conclude that PSaV particles enter LLC-PK cells by binding to occludin as a coreceptor in PSaV-dissociated TJs. PSaV and occludin then form a complex that moves to late endosomes via Rab5- and Rab7-dependent trafficking. In addition, claudin-1 in the TJs opened by PSaV infection facilitates PSaV entry and infection as an entry factor. Sapoviruses (SaVs) cause severe acute gastroenteritis in humans and animals. Although they replicate in intestinal epithelial cells, which are tightly sealed by apical-junctional complexes, such as tight junctions (TJs), the mechanisms by which SaVs hijack TJs and their proteins for successful entry and infection remain largely unknown. Here, we demonstrate that porcine SaVs (PSaVs) induce early dissociation of TJs, allowing them to bind to the TJ protein occludin as a functional coreceptor. PSaVs then travel in a complex with occludin into late endosomes through Rab5- and Rab7-dependent trafficking. Claudin-1, another TJ protein, does not directly interact with PSaV but facilitates the entry of PSaV into cells as an entry factor. This work contributes to our understanding of the entry of SaV and other caliciviruses into cells and may aid in the development of efficient and affordable drugs to treat SaV infections.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1128/JVI.01773-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6364031PMC
February 2019

Phosphatidylinositol 3-Kinase/Akt and MEK/ERK Signaling Pathways Facilitate Sapovirus Trafficking and Late Endosomal Acidification for Viral Uncoating in LLC-PK Cells.

J Virol 2018 12 27;92(24). Epub 2018 Nov 27.

Laboratory of Veterinary Pathology, College of Veterinary Medicine, Chonnam National University, Gwangju, Republic of Korea

Sapovirus, an important cause of acute gastroenteritis in humans and animals, travels from the early to the late endosomes and requires late endosomal acidification for viral uncoating. However, the signaling pathways responsible for these viral entry processes remain unknown. Here we demonstrate the receptor-mediated early activation of phosphatidylinositol 3-kinase (PI3K)/Akt and mitogen-activated protein extracellular signal-regulated kinase/extracellular signal-regulated kinase (MEK/ERK) signaling pathways involved in sapovirus entry processes. Both signaling pathways were activated during the early stage of porcine sapovirus (PSaV) infection. However, depletion of the cell surface carbohydrate receptors by pretreatment with sodium periodate or neuraminidase reduced the PSaV-induced early activation of these signaling pathways, indicating that PSaV binding to the cell surface carbohydrate receptors triggered these cascades. Addition of bile acid, known to be essential for PSaV escape from late endosomes, was also found to exert a stiffening effect to stimulate both pathways. Inhibition of these signaling pathways by use of inhibitors specific for PI3K or MEK or small interfering RNAs (siRNAs) against PI3K or MEK resulted in entrapment of PSaV particles in early endosomes and prevented their trafficking to late endosomes. Moreover, phosphorylated PI3K and ERK coimmunoprecipitated subunit E of the V-ATPase proton pump that is important for endosomal acidification. Based on our data, we conclude that receptor binding of PSaV activates both PI3K/Akt and MEK/ERK signaling pathways, which in turn promote PSaV trafficking from early to late endosomes and acidification of late endosomes for PSaV uncoating. These signaling cascades may provide a target for potent therapeutics against infections by PSaV and other caliciviruses. Sapoviruses cause acute gastroenteritis in both humans and animals. However, the host signaling pathway(s) that facilitates host cell entry by sapoviruses remains largely unknown. Here we demonstrate that porcine sapovirus (PSaV) activates both PI3K/Akt and MEK/ERK cascades at an early stage of infection. Removal of cell surface receptors decreased PSaV-induced early activation of both cascades. Moreover, blocking of PI3K/Akt and MEK/ERK cascades entrapped PSaV particles in early endosomes and prevented their trafficking to the late endosomes. PSaV-induced early activation of PI3K and ERK molecules further mediated V-ATPase-dependent late endosomal acidification for PSaV uncoating. This work unravels a new mechanism by which receptor-mediated early activation of both cascades may facilitate PSaV trafficking from early to late endosomes and late endosomal acidification for PSaV uncoating, which in turn can be a new target for treatment of sapovirus infection.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1128/JVI.01674-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6258943PMC
December 2018

Rotavirus-Induced Early Activation of the RhoA/ROCK/MLC Signaling Pathway Mediates the Disruption of Tight Junctions in Polarized MDCK Cells.

Sci Rep 2018 09 17;8(1):13931. Epub 2018 Sep 17.

Laboratory of Veterinary Pathology, College of Veterinary Medicine, Chonnam National University, Gwangju, Republic of Korea.

Intestinal epithelial tight junctions (TJ) are a major barrier restricting the entry of various harmful factors including pathogens; however, they also represent an important entry portal for pathogens. Although the rotavirus-induced early disruption of TJ integrity and targeting of TJ proteins as coreceptors are well-defined, the precise molecular mechanisms involved remain unknown. In the present study, infection of polarized MDCK cells with the species A rotavirus (RVA) strains human DS-1 and bovine NCDV induced a redistribution of TJ proteins into the cytoplasm, a reversible decrease in transepithelial resistance, and an increase in paracellular permeability. RhoA/ROCK/MLC signaling was identified as activated at an early stage of infection, while inhibition of this pathway prevented the rotavirus-induced early disruption of TJ integrity and alteration of TJ protein distribution. Activation of pMYPT, PKC, or MLCK, which are known to participate in TJ dissociation, was not observed in MDCK cells infected with either rotavirus strain. Our data demonstrated that binding of RVA virions or cogent VP8* proteins to cellular receptors activates RhoA/ROCK/MLC signaling, which alters TJ protein distribution and disrupts TJ integrity via contraction of the perijunctional actomyosin ring, facilitating virion access to coreceptors and entry into cells.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41598-018-32352-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6141481PMC
September 2018

Porcine sapovirus Cowden strain enters LLC-PK cells via clathrin- and cholesterol-dependent endocytosis with the requirement of dynamin II.

Vet Res 2018 Sep 17;49(1):92. Epub 2018 Sep 17.

Laboratory of Veterinary Pathology, College of Veterinary Medicine, Chonnam National University, Gwangju, Republic of Korea.

Caliciviruses in the genus Sapovirus are a significant cause of viral gastroenteritis in humans and animals. However, the mechanism of their entry into cells is not well characterized. Here, we determined the entry mechanism of porcine sapovirus (PSaV) strain Cowden into permissive LLC-PK cells. The inhibition of clathrin-mediated endocytosis using chlorpromazine, siRNAs, and a dominant negative (DN) mutant blocked entry and infection of PSaV Cowden strain, confirming a role for clathrin-mediated internalization. Entry and infection were also inhibited by the cholesterol-sequestering drug methyl-β-cyclodextrin and was restored by the addition of soluble cholesterol, indicating that cholesterol also contributes to entry and infection of this strain. Furthermore, the inhibition of dynamin GTPase activity by dynasore, siRNA depletion of dynamin II, or overexpression of a DN mutant of dynamin II reduced the entry and infection, suggesting that dynamin mediates the fission and detachment of clathrin- and cholesterol-pits for entry of this strain. In contrast, the inhibition of caveolae-mediated endocytosis using nystatin, siRNAs, or a DN mutant had no inhibitory effect on entry and infection of this strain. It was further determined that cell entry of PSaV Cowden strain required actin rearrangements for vesicle internalization, endosomal trafficking from early to late endosomes through microtubules, and late endosomal acidification for uncoating. We conclude that PSaV strain Cowden is internalized into LLC-PK cells by clathrin- and cholesterol-mediated endocytosis that requires dynamin II and actin rearrangement, and that the uncoating occurs in the acidified late endosomes after trafficking from the early endosomes through microtubules.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/s13567-018-0584-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6142377PMC
September 2018

Feline calicivirus- and murine norovirus-induced COX-2/PGE2 signaling pathway has proviral effects.

PLoS One 2018 18;13(7):e0200726. Epub 2018 Jul 18.

Laboratory of Veterinary Pathology, College of Veterinary Medicine, Chonnam National University, Gwangju, Republic of Korea.

Cyclooxygenases (COXs)/prostaglandin E2 (PGE2) signaling pathways are known to modulate a variety of homeostatic processes and are involved in various pathophysiological conditions. COXs/PGE2 signaling pathways have also been demonstrated to have proviral or antiviral effects, which appeared different even in the same virus family. A porcine sapovirus Cowden strain, a member of genus Sapovirus within the Caliciviridae family, induces strong COX-2/PGE2 but transient COX-1/PGE2 signaling to enhance virus replication. However, whether infections of other viruses in the different genera activate COXs/PGE2 signaling, and thus affect the replication of viruses, remains unknown. In the present study, infections of cells with the feline calicivirus (FCV) F9 strain in the genus Vesivirus and murine norovirus (MNV) CW-1 strain in the genus Norovirus only activated the COX-2/PGE2 signaling in a time-dependent manner. Treatment with pharmacological inhibitors or transfection of small interfering RNAs (siRNAs) against COX-2 enzyme significantly reduced the production of PGE2 as well as FCV and MNV replications. The inhibitory effects of these pharmacological inhibitors against COX-2 enzyme on the replication of both viruses were restored by the addition of PGE2. Silencing of COX-1 via siRNAs and inhibition of COX-1 via an inhibitor also decrease the production of PGE2 and replication of both viruses, which can be attributed to the inhibition COX-1/PGE2 signaling pathway. These data indicate that the COX-2/PGE2 signaling pathway has proviral effects for the replication of FCV and MNV, and pharmacological inhibitors against these enzymes serve as potential therapeutic candidates for treating FCV and MNV infections.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0200726PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6051663PMC
January 2019

Bovine Nebovirus Interacts with a Wide Spectrum of Histo-Blood Group Antigens.

J Virol 2018 05 13;92(9). Epub 2018 Apr 13.

Laboratory of Veterinary Pathology, College of Veterinary Medicine, Chonnam National University, Gwangju, Republic of Korea

Some viruses within the family initiate their replication cycle by attachment to cell surface carbohydrate moieties, histo-blood group antigens (HBGAs), and/or terminal sialic acids (SAs). Although bovine nebovirus (BNeV), one of the enteric caliciviruses, is an important causative agent of acute gastroenteritis in cattle, its attachment factors and possibly other cellular receptors remain unknown. Using a comprehensive series of protein-ligand biochemical assays, we sought to determine whether BNeV recognizes cell surface HBGAs and/or SAs as attachment factors. It was found that BNeV virus-like particles (VLPs) bound to A type/H type 2/Le HBGAs expressed in the bovine digestive tract and are related to HBGAs expressed in humans and other host species, suggesting a wide spectrum of HBGA recognition by BNeV. BNeV VLPs also bound to a large variety of different bovine and human saliva samples of all ABH and Lewis types, supporting previously obtained results and suggesting a zoonotic potential of BNeV transmission. Removal of α1,2-linked fucose and α1,3/4-linked fucose epitopes of target HBGAs by confirmation-specific enzymes reduced the binding of BNeV VLPs to synthetic HBGAs, bovine and human saliva, cultured cell lines, and bovine small intestine mucosa, further supporting a wide HBGA binding spectrum of BNeV through recognition of α1,2-linked fucose and α1,3/4-linked fucose epitopes of targeted HBGAs. However, removal of terminal α2,3- and α2,6-linked SAs by their specific enzyme had no inhibitory effects on binding of BNeV VLPs, indicating that BNeV does not use terminal SAs as attachment factors. Further details of the binding specificity of BNeV remain to be explored. Enteric caliciviruses such as noroviruses, sapoviruses, and recoviruses are the most important etiological agents of severe acute gastroenteritis in humans and many other mammalian host species. They initiate infection by attachment to cell surface carbohydrate moieties, HBGAs, and/or terminal SAs. However, the attachment factor(s) for BNeV, a recently classified enteric calicivirus genus/type species, remains unexplored. Here, we demonstrate that BNeV VLPs have a wide spectrum of binding to synthetic HBGAs, bovine and human saliva samples, and bovine duodenal sections. We further discovered that α1,2-linked fucose and α1,3/4-linked fucose epitopes are essential for binding of BNeV VLPs. However, BNeV VLPs do not bind to terminal SAs on cell carbohydrates. Continued investigation regarding the proteinaceous receptor(s) will be necessary for better understanding of the tropism, pathogenesis, and host range of this important viral genus.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1128/JVI.02160-17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5899197PMC
May 2018

Activation of PI3K, Akt, and ERK during early rotavirus infection leads to V-ATPase-dependent endosomal acidification required for uncoating.

PLoS Pathog 2018 01 19;14(1):e1006820. Epub 2018 Jan 19.

Laboratory of Veterinary Pathology, College of Veterinary Medicine, Chonnam National University, Gwangju, Republic of Korea.

The cellular PI3K/Akt and/or MEK/ERK signaling pathways mediate the entry process or endosomal acidification during infection of many viruses. However, their roles in the early infection events of group A rotaviruses (RVAs) have remained elusive. Here, we show that late-penetration (L-P) human DS-1 and bovine NCDV RVA strains stimulate these signaling pathways very early in the infection. Inhibition of both signaling pathways significantly reduced production of viral progeny due to blockage of virus particles in the late endosome, indicating that neither of the two signaling pathways is involved in virus trafficking. However, immunoprecipitation assays using antibodies specific for pPI3K, pAkt, pERK and the subunit E of the V-ATPase co-immunoprecipitated the V-ATPase in complex with pPI3K, pAkt, and pERK. Moreover, Duolink proximity ligation assay revealed direct association of the subunit E of the V-ATPase with the molecules pPI3K, pAkt, and pERK, indicating that both signaling pathways are involved in V-ATPase-dependent endosomal acidification. Acidic replenishment of the medium restored uncoating of the RVA strains in cells pretreated with inhibitors specific for both signaling pathways, confirming the above results. Isolated components of the outer capsid proteins, expressed as VP4-VP8* and VP4-VP5* domains, and VP7, activated the PI3K/Akt and MEK/ERK pathways. Furthermore, psoralen-UV-inactivated RVA and CsCl-purified RVA triple-layered particles triggered activation of the PI3K/Akt and MEK/ERK pathways, confirming the above results. Our data demonstrate that multistep binding of outer capsid proteins of L-P RVA strains with cell surface receptors phosphorylates PI3K, Akt, and ERK, which in turn directly interact with the subunit E of the V-ATPase to acidify the late endosome for uncoating of RVAs. This study provides a better understanding of the RVA-host interaction during viral uncoating, which is of importance for the development of strategies aiming at controlling or preventing RVA infections.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1371/journal.ppat.1006820DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5792019PMC
January 2018

Use of computed tomography and minimum intensity projection in the detection of lobar pneumonia mimicking lung lobe torsion in a dog.

Vet Radiol Ultrasound 2019 Sep 16;60(5):E48-E53. Epub 2017 Oct 16.

College of Veterinary Medicine and BK 21 Plus Project Team, Chonnam National University, Gwangju, 61186, South Korea.

A 10-year-old female spayed Dachshund was referred with progressive coughing for 1 month. The dog was tentatively diagnosed with right middle lung torsion based on pleural effusion, vesicular emphysema, abruptly ending bronchus in consolidated right middle lung, and no contrast enhancement of the affected lobe on radiography and computed tomography (CT). There was no evidence of torsion upon thoracotomy, and histological examination confirmed lobar pneumonia. The CT images were reevaluated using minimum intensity projection and revealed normal bronchial courses. The minimum intensity projection technique can be to assist in evaluation of the bronchial tree for dogs with suspected lung lobe torsion and other pulmonary diseases.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/vru.12565DOI Listing
September 2019

Glycan-specificity of four neuraminidase-sensitive animal rotavirus strains.

Vet Microbiol 2017 Aug 23;207:159-163. Epub 2017 Jun 23.

Laboratory of Veterinary Pathology, College of Veterinary Medicine, Chonnam National University, Gwangju 61186, Republic of Korea. Electronic address:

Group A rotaviruses (RVAs) are divided into neuraminidase (NA)-sensitive and NA-insensitive strains depending upon their binding affinity to the VP8* domain in the terminal sialic acids (SAs) of cell surface carbohydrates. Although NA-sensitive strains are known to use terminal SAs as an attachment factor, the exact nature of this attachment factor is largely unknown. Here we show that the specific linkage of SA-containing glycan to glycoprotein or glycolipid is an attachment factor used by NA-sensitive porcine G9P[7] PRG9121 and G9P[23] PRG942, bovine G6P[1] NCDV, and canine G3P[3] strains. Infectivity of porcine G9P[7] and G9P[23] strains was markedly blocked by α2,3-linkage and α2,6-linkage inhibitors, indicating that these strains bind to both α2,3- and α2,6-linked SAs. However, the infectivity of bovine G6P[1] and canine G3P[3] strains was significantly reduced by α2,6-linkage inhibitor but not by α2,3-linkage blockers, demonstrating a predilection of these strains for α2,6-linked SAs. The infectivity of four NA-sensitive strains was equally reduced by inhibitors of lipid membrane and N-linked glycoprotein but not by an inhibitor of O-linked glycoprotein, indicating that these strains utilize both glycolipid and N-linked glycoprotein. Our study demonstrates that four NA-sensitive animal strains could have a strain-dependent binding preference toward α2,6-linked SAs (P[1] NCDV and P[3] CU-1 strains) or both α2,3- and α2,6-linked SAs (P[7] PRG9121 and P[23] PRG942 strains) to the glycolipid and N-linked glycoprotein.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.vetmic.2017.06.016DOI Listing
August 2017

Comparison of elastography, contrast-enhanced ultrasonography, and computed tomography for assessment of lesion margin after radiofrequency ablation in livers of healthy dogs.

Am J Vet Res 2017 Mar;78(3):295-304

OBJECTIVE To assess by use of various diagnostic imaging modalities acute changes in livers of healthy dogs after radiofrequency ablation (RFA) and determine the capability of each imaging modality to monitor ablation lesion changes. ANIMALS 6 healthy Beagles. PROCEDURES 12 ablation lesions were created in the liver of the dogs (2 lesions/dog). Ablation lesions were evaluated by use of conventional ultrasonography, strain elastography, and contrast-enhanced ultrasonography immediately after (time 0), 30 to 60 minutes after, and 3 days after RFA, and by use of CT 30 minutes and 3 days after RFA. Three dogs were euthanized shortly after RFA, and the other 3 dogs were euthanized on day 3. Lesion size measured by each imaging modality was compared with necropsy findings. RESULTS Immediately after RFA, clear margins were more visible with elastography and contrast-enhanced ultrasonography than with conventional ultrasonography, which had acoustic shadowing. On triphasic contrast CT, the ablation zone, which indicated necrosis and hemorrhage, was not enhanced and could be measured. Marked enhancement of the periablation rim was observed during the venous phase and was identified as granulation tissue. Size of the ablation area measured on enhanced CT images was strongly correlated with actual lesion size. CONCLUSIONS AND CLINICAL RELEVANCE For dogs of this study, CT was the most reliable method for lesion size determination. Although ultrasonographic imaging measurements underestimated lesion size, all modalities could be used to provide additional real-time guidance for RFA procedures of the liver as well as for other RFA procedures.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.2460/ajvr.78.3.295DOI Listing
March 2017

Activation of COX-2/PGE2 Promotes Sapovirus Replication via the Inhibition of Nitric Oxide Production.

J Virol 2017 Feb 18;91(3). Epub 2017 Jan 18.

Laboratory of Veterinary Pathology, College of Veterinary Medicine, Chonnam National University, Gwangju, Republic of Korea

Enteric caliciviruses in the genera Norovirus and Sapovirus are important pathogens that cause severe acute gastroenteritis in both humans and animals. Cyclooxygenases (COXs) and their final product, prostaglandin E (PGE), are known to play important roles in the modulation of both the host response to infection and the replicative cycles of several viruses. However, the precise mechanism(s) by which the COX/PGE pathway regulates sapovirus replication remains largely unknown. In this study, infection with porcine sapovirus (PSaV) strain Cowden, the only cultivable virus within the genus Sapovirus, markedly increased COX-2 mRNA and protein levels at 24 and 36 h postinfection (hpi), with only a transient increase in COX-1 levels seen at 24 hpi. The treatment of cells with pharmacological inhibitors, such as nonsteroidal anti-inflammatory drugs or small interfering RNAs (siRNAs) against COX-1 and COX-2, significantly reduced PGE production, as well as PSaV replication. Expression of the viral proteins VPg and ProPol was associated with activation of the COX/PGE pathway. We observed that pharmacological inhibition of COX-2 dramatically increased NO production, causing a reduction in PSaV replication that could be restored by inhibition of nitric oxide synthase via the inhibitor N-nitro-l-methyl-arginine ester. This study identified a pivotal role for the COX/PGE pathway in the regulation of NO production during the sapovirus life cycle, providing new insights into the life cycle of this poorly characterized family of viruses. Our findings also reveal potential new targets for treatment of sapovirus infection.

Importance: Sapoviruses are among the major etiological agents of acute gastroenteritis in both humans and animals, but little is known about sapovirus host factor requirements. Here, using only cultivable porcine sapovirus (PSaV) strain Cowden, we demonstrate that PSaV induced the vitalization of the cyclooxygenase (COX) and prostaglandin E (PGE) pathway. Targeting of COX-1/2 using nonsteroidal anti-inflammatory drugs (NSAIDs) such as the COX-1/2 inhibitor indomethacin and the COX-2-specific inhibitors NS-398 and celecoxib or siRNAs targeting COXs, inhibited PSaV replication. Expression of the viral proteins VPg and ProPol was associated with activation of the COX/PGE pathway. We further demonstrate that the production of PGE provides a protective effect against the antiviral effector mechanism of nitric oxide. Our findings uncover a new mechanism by which PSaV manipulates the host cell to provide an environment suitable for efficient viral growth, which in turn can be a new target for treatment of sapovirus infection.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1128/JVI.01656-16DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5244346PMC
February 2017

Occurrence and molecular characterization of Sapelovirus A in diarrhea and non-diarrhea feces of different age group pigs in one Korean pig farm.

J Vet Med Sci 2017 Jan 11;78(12):1911-1914. Epub 2016 Sep 11.

Laboratory of Veterinary Pathology, College of Veterinary Medicine, Chonnam National University, Gwangju, Republic of Korea.

To determine the occurrence and genetic diversity of Sapelovirus A (SV-A) in diarrhea and non-diarrhea feces of Korean pigs, 110 specimens from different age groups of pigs in the same farm were analyzed by RT-nested PCR. SV-As were detected in 60% of both diarrhea and non-diarrhea specimens regardless of age groups with primer pairs for 2C region, in which all diarrhea samples were co-infected by other enteric pathogens. Phylogenetical analysis of partial VP1 region showed that our strains and several other Korean strains belonged to cluster I, distinct from some strains reported in Korea and other countries. These data indicate that genetically distinct SV-As are frequently detected in Korean pigs irrespective of diarrhea and age.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1292/jvms.16-0237DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5240775PMC
January 2017

Pathogenesis of Korean SapelovirusA in piglets and chicks.

J Gen Virol 2016 10 2;97(10):2566-2574. Epub 2016 Aug 2.

Laboratory of Veterinary Pathology, College of Veterinary Medicine, Chonnam National University, Gwangju, Republic of Korea.

Sapelovirus A (SV-A), formerly known as porcine sapelovirus as a member of a new genus Sapelovirus, is known to cause enteritis, pneumonia, polioencephalomyelitis and reproductive disorders in pigs. We have recently identified α2,3-linked sialic acid on GD1a ganglioside as a functional SV-A receptor rich in the cells of pigs and chickens. However, the role of GD1a in viral pathogenesis remains elusive. Here, we demonstrated that a Korean SV-A strain could induce diarrhoea and intestinal pathology in piglets but not in chicks. Moreover, this Korean SV-A strain had mild extra-intestinal tropisms appearing as mild, non-suppurative myelitis, encephalitis and pneumonia in piglets, but not in chicks. By real-time reverse transcription (RT) PCR, higher viral RNA levels were detected in faecal samples than in sera or extra-intestinal organs from virus-inoculated piglets. Immunohistochemistry confirmed that high viral antigens were detected in the epithelial cells of intestines from virus-inoculated piglets but not from chicks. This Korean SV-A strain could bind the cultured cell lines originated from various species, but replication occurred only in cells of porcine origin. These data indicated that this Korean SV-A strain could replicate and induce pathology in piglets but not in chicks, suggesting that additional porcine-specific factors are required for virus entry and replication. In addition, this Korean SV-A strain is enteropathogenic, but could spread to the bloodstream from the gut and disseminate to extra-intestinal organs and tissues. These results will contribute to our understanding of SV-A pathogenesis so that efficient anti-sapelovirus drugs and vaccines could be developed in the future.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1099/jgv.0.000571DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5078829PMC
October 2016