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Canine natural killer (NK) cells are large, granular lymphocytes that are neither B lymphocytes nor T lymphocytes. However, it has been reported that canine NK cells share some of the phenotypic characteristics of T lymphocytes, such as CD3 and CD5. Studies are needed to assess the safety of canine NK cells for immunotherapy, especially because the safety of using allogeneic NK cells as an immunotherapy for dogs has yet to be shown. In this study, the safety of cultured canine NK cells was assessed using a xenogeneic mouse model of graft-versus-host disease (GVHD). Mice were injected with either canine peripheral blood mononuclear cells (PBMCs) or cultured NK cells for 2 or 3 weeks. Data were then collected on changes in mice body weights, disease severity scores, and survival rates. Histopathological and immunohistochemical evaluations were also performed. All mice injected with canine PBMCs died within 45 days after injection. Severe clinical signs were caused by GVHD. The histopathological and immunohistochemical evaluations showed that mice injected with canine PBMCs had multiple lesions, including necrosis in their lungs, livers, kidneys, and stomachs, and the injected cells were present around the lesions. By contrast, no mice injected with cultured NK cells without removing the CD3+TCR– cells exhibited any clinical abnormalities. Moreover, they all survived the 90-day experimental period without exhibiting any histopathological changes. Accordingly, the results of this study suggest that canine NK cells do not cause significant side effects such as GVHD and allogeneic NK cells can safely be used for cancer immunotherapy in dogs.

Group A rotavirus (RVA), one of the leading pathogens causing severe acute gastroenteritis in children and a wide variety of young animals worldwide, induces apoptosis upon infecting cells. Though RVA-induced apoptosis mediated via the dual modulation of its NSP4 and NSP1 proteins is relatively well studied, the nature and signaling pathway(s) involved in RVA-induced necroptosis are yet to be fully elucidated. Here, we demonstrate the nature of RVA-induced necroptosis, the signaling cascade involved, and correlation with RVA-induced apoptosis. Infection with the bovine NCDV and human DS-1 RVA strains was shown to activate receptor-interacting protein kinase 1 (RIPK1), RIPK3, and mixed-lineage kinase domain-like protein (MLKL), the key necroptosis molecules in virus-infected cells. Using an immunoprecipitation assay, RIPK1 was found to bind phosphorylated RIPK3 (pRIPK3) and pMLKL. pMLKL, the major executioner molecule in the necroptotic pathway, was translocated to the plasma membrane of RVA-infected cells to puncture the cell membrane. Interestingly, transfection of RVA NSP4 also induced necroptosis through the RIPK1/RIPK3/MLKL necroptosis pathway. Blockage of each key necroptosis molecule in the RVA-infected or NSP4-transfected cells resulted in decreased necroptosis but increased cell viability and apoptosis, thereby resulting in decreased viral yields in the RVA-infected cells. In contrast, suppression of RVA-induced apoptosis increased necroptosis and virus yields. Our findings suggest that RVA NSP4 also induces necroptosis via the RIPK1/RIPK3/MLKL necroptosis pathway. Moreover, necroptosis and apoptosis—which have proviral and antiviral effects, respectively—exhibited cross talk in RVA-infected cells. These findings significantly increase our understanding of the nature of RVA-induced necroptosis and the cross talk between RVA-induced necroptosis and apoptosis. IMPORTANCE Viral infection usually culminates in cell death through apoptosis, necroptosis, and, rarely, pyroptosis. Necroptosis is a form of programmed necrosis that is mediated by signaling complexes of the receptor-interacting protein kinase 1 (RIPK1), RIPK3, and mixed-lineage kinase domain-like protein (MLKL). Although apoptosis induction by rotavirus and its NSP4 protein is well known, rotavirus-induced necroptosis is not fully understood. Here, we demonstrate that rotavirus and also its NSP4 protein can induce necroptosis in cultured cells through activation of the RIPK1/RIPK3/MLKL necroptosis pathway. Moreover, rotavirus-induced necroptosis and apoptosis have opposite effects on viral yield, i.e., they function as proviral and antiviral processes, respectively, and counterbalance each other in rotavirus-infected cells. Our findings provide important insights for understanding the nature of rotavirus-induced necroptosis and the development of novel therapeutic strategies against infection with rotavirus and other RNA viruses.

18 Pág. Centro de Biotecnología y Genómica de Plantas (CBGP), Recombinant viruses expressing reporter genes allow visualization and quantification of viral infections and can be used as valid surrogates to identify the presence of the virus in infected cells and animal models. However, one of the limitations of recombinant viruses expressing reporter genes is the use of either fluorescent or luciferase proteins that are used alternatively for different purposes. Vaccinia virus (VV) is widely used as a viral vector, including recombinant (r)VV singly expressing either fluorescent or luciferase reporter genes that are useful for specific purposes. In this report, we engineered two novel rVV stably expressing both fluorescent (Scarlet or GFP) and luciferase (Nluc) reporter genes from different loci in the viral genome. In vitro, these bi-reporter-expressing rVV have similar growth kinetics and plaque phenotype than those of the parental WR VV isolate. In vivo, rVV Nluc/Scarlet and rVV Nluc/GFP effectively infected mice and were easily detected using in vivo imaging systems (IVIS) and ex vivo in the lungs from infected mice. Importantly, we used these bi-reporter-expressing rVV to assess viral pathogenesis, infiltration of immune cells in the lungs, and to directly identify the different subsets of cells infected by VV in the absence of antibody staining. Collectively, these rVV expressing two reporter genes open the feasibility to study the biology of viral infections in vitro and in vivo, including host-pathogen interactions and dynamics or tropism of viral infections. IMPORTANCE Despite the eradication of variola virus (VARV), the causative agent of smallpox, poxviruses still represent an important threat to human health due to their possible use as bioterrorism agents and the emergence of zoonotic poxvirus diseases. Recombinant vaccinia viruses (rVV) expressing easily traceable fluorescent or luciferase reporter genes have significantly contributed to the progress of poxvirus research. However, rVV expressing one marker gene have several constraints for in vitro and in vivo studies, since both fluorescent and luciferase proteins impose certain limitations for specific applications. To overcome these limitations, we generated optimized rVV stably expressing both fluorescent (Scarlet or GFP) and luciferase (Nluc) reporter genes to easily track viral infection in vitro and in vivo. This new generation of double reporter-expressing rVV represent an excellent option to study viral infection dynamics in cultured cells and validated animal models of infection., This work was supported by grants E-RTA2014-00006, RTA2017-0066 from Ministerio de Economía y Competitividad and Ministerio de Ciencia, Innovación y Universidades as part of the Plan Estatal de Investigación Científica, Desarrollo e Innovación Tecnológica, and grant COV20-00901 from Instituto de Salud Carlos III (ISCIII)

The coronavirus disease 2019 (COVID-19) pandemic has been caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). To date, SARS-CoV-2 has been responsible for over 242 million infections and more than 4.9 million deaths worldwide. Similar to other viruses, studying SARS-CoV-2 requires the use of experimental methods to detect the presence of virus in infected cells and/or in animal models. To overcome this limitation, we generated replication-competent recombinant (r)SARS-CoV-2 that expresses bioluminescent (nanoluciferase, Nluc) or fluorescent (Venus) proteins. These reporter-expressing rSARS-CoV-2 allow tracking viral infections in vitro and in vivo based on the expression of Nluc and Venus reporter genes. Here the study describes the use of rSARS-CoV-2/Nluc and rSARS-CoV-2/Venus to detect and track SARS-CoV-2 infection in the previously described K18 human angiotensin-converting enzyme 2 (hACE2) transgenic mouse model of infection using in vivo imaging systems (IVIS). This rSARS-CoV-2/Nluc and rSARS-CoV-2/Venus show rSARS-CoV-2/WT-like pathogenicity and viral replication in vivo. Importantly, Nluc and Venus expression allow us to directly track viral infections in vivo and ex vivo, in infected mice. These rSARS-CoV-2/Nluc and rSARS-CoV-2/Venus represent an excellent option to study the biology of SARS-CoV-2 in vivo, to understand viral infection and associated COVID-19 disease, and to identify effective prophylactic and/or therapeutic treatments to combat SARS-CoV-2 infection.

A comprehensive analysis and characterization of a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection model that mimics non-severe and severe coronavirus disease 2019 (COVID-19) in humans is warranted for understating the virus and developing preventive and therapeutic agents. Here, we characterized the K18-hACE2 mouse model expressing human (h)ACE2 in mice, controlled by the human keratin 18 (K18) promoter, in the epithelia, including airway epithelial cells where SARS-CoV-2 infections typically start. We found that intranasal inoculation with higher viral doses (2 × 103 and 2 × 104 PFU) of SARS-CoV-2 caused lethality of all mice and severe damage of various organs, including lung, liver, and kidney, while lower doses (2 × 101 and 2 × 102 PFU) led to less severe tissue damage and some mice recovered from the infection. In this hACE2 mouse model, SARS-CoV-2 infection damaged multiple tissues, with a dose-dependent effect in most tissues. Similar damage was observed in postmortem samples from COVID-19 patients. Finally, the mice that recovered from infection with a low dose of virus survived rechallenge with a high dose of virus. Compared to other existing models, the K18-hACE2 model seems to be the most sensitive COVID-19 model reported to date. Our work expands the information available about this model to include analysis of multiple infectious doses and various tissues with comparison to human postmortem samples from COVID-19 patients. In conclusion, the K18-hACE2 mouse model recapitulates both severe and non-severe COVID-19 in humans being dose-dependent and can provide insight into disease progression and the efficacy of therapeutics for preventing or treating COVID-19. IMPORTANCE The pandemic of coronavirus disease 2019 (COVID-19) has reached nearly 240 million cases, caused nearly 5 million deaths worldwide as of October 2021, and has raised an urgent need for the development of novel drugs and therapeutics to prevent the spread and pathogenesis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). To achieve this goal, an animal model that recapitulates the features of human COVID-19 disease progress and pathogenesis is greatly needed. In this study, we have comprehensively characterized a mouse model of SARS-CoV-2 infection using K18-hACE2 transgenic mice. We infected the mice with low and high doses of SARS-CoV-2 to study the pathogenesis and survival in response to different infection patterns. Moreover, we compared the pathogenesis of the K18-hACE2 transgenic mice with that of the COVID-19 patients to show that this model could be a useful tool for the development of antiviral drugs and therapeutics.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged at the end of 2019 and has been responsible for the still ongoing coronavirus disease 2019 (COVID-19) pandemic. Prophylactic vaccines have been authorized by the U.S. Food and Drug Administration (FDA) for the prevention of COVID-19. Identification of SARS-CoV-2-neutralizing antibodies (NAbs) is important to assess vaccine protection efficacy, including their ability to protect against emerging SARS-CoV-2 variants of concern (VoC). Here, we report the generation and use of a recombinant (r)SARS-CoV-2 USA/WA1/2020 (WA-1) strain expressing Venus and an rSARS-CoV-2 strain expressing mCherry and containing mutations K417N, E484K, and N501Y found in the receptor binding domain (RBD) of the spike (S) glycoprotein of the South African (SA) B.1.351 (beta [β]) VoC in bifluorescent-based assays to rapidly and accurately identify human monoclonal antibodies (hMAbs) able to neutralize both viral infections in vitro and in vivo. Importantly, our bifluorescent-based system accurately recapitulated findings observed using individual viruses. Moreover, fluorescent-expressing rSARS-CoV-2 strain and the parental wild-type (WT) rSARS-CoV-2 WA-1 strain had similar viral fitness in vitro, as well as similar virulence and pathogenicity in vivo in the K18 human angiotensin-converting enzyme 2 (hACE2) transgenic mouse model of SARS-CoV-2 infection. We demonstrate that these new fluorescent-expressing rSARS-CoV-2 can be used in vitro and in vivo to easily identify hMAbs that simultaneously neutralize different SARS-CoV-2 strains, including VoC, for the rapid assessment of vaccine efficacy or the identification of prophylactic and/or therapeutic broadly NAbs for the treatment of SARS-CoV-2 infection. IMPORTANCE SARS-CoV-2 is responsible of the COVID-19 pandemic that has warped daily routines and socioeconomics. There is still an urgent need for prophylactics and therapeutics to treat SARS-CoV-2 infections. In this study, we demonstrate the feasibility of using bifluorescent-based assays for the rapid identification of hMAbs with neutralizing activity against SARS-CoV-2, including VoC in vitro and in vivo. Importantly, results obtained with these bifluorescent-based assays recapitulate those observed with individual viruses, demonstrating their feasibility to rapidly advance our understanding of vaccine efficacy and to identify broadly protective human NAbs for the therapeutic treatment of SARS-CoV-2.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of the current COVID-19 pandemic, is one of the biggest threats to public health. However, the dynamic of SARS-CoV-2 infection remains poorly understood. Replication-competent recombinant viruses expressing reporter genes provide valuable tools to investigate viral infection. Low levels of reporter gene expressed from previous reporter-expressing recombinant (r)SARS-CoV-2 in the locus of the open reading frame (ORF)7a protein have jeopardized their use to monitor the dynamic of SARS-CoV-2 infection in vitro or in vivo. Here, we report an alternative strategy where reporter genes were placed upstream of the highly expressed viral nucleocapsid (N) gene followed by a porcine tescherovirus (PTV-1) 2A proteolytic cleavage site. The higher levels of reporter expression using this strategy resulted in efficient visualization of rSARS-CoV-2 in infected cultured cells and excised lungs or whole organism of infected K18 human angiotensin converting enzyme 2 (hACE2) transgenic mice. Importantly, real-time viral infection was readily tracked using a noninvasive in vivo imaging system and allowed us to rapidly identify antibodies which are able to neutralize SARS-CoV-2 infection in vivo. Notably, these reporter-expressing rSARS-CoV-2, in which a viral gene was not deleted, not only retained wild-type (WT) virus-like pathogenicity in vivo but also exhibited high stability in vitro and in vivo, supporting their use to investigate viral infection, dissemination, pathogenesis, and therapeutic interventions for the treatment of SARS-CoV-2 in vivo.

Recombinant viruses expressing reporter genes allow visualization and quantification of viral infections and can be used as valid surrogates to identify the presence of the virus in infected cells and animal models. However, one of the limitations of recombinant viruses expressing reporter genes is the use of either fluorescent or luciferase proteins that are used alternatively for different purposes. Vaccinia virus (VV) is widely used as a viral vector, including recombinant (r)VV singly expressing either fluorescent or luciferase reporter genes that are useful for specific purposes. In this report, we engineered two novel rVV stably expressing both fluorescent (Scarlet or GFP) and luciferase (Nluc) reporter genes from different loci in the viral genome. In vitro, these bi-reporter expressing rVV have similar growth kinetics and plaque phenotype than those of the parental WR VV isolate. In vivo, rVV Nluc/Scarlet and rVV Nluc/GFP effectively infected mice and were easily detected using in vivo imaging systems (IVIS) and ex vivo in the lungs from infected mice. We used these bi-reporter expressing rVV to assess viral pathogenesis, infiltration of immune cells in the lungs, and to directly identify the different subsets of cells infected by VV in the absence of antibody staining. Collectively, these rVV expressing two reporter genes open the feasibility to study the biology of viral infections in vitro and in vivo, including host-pathogen interactions and dynamics or tropism of viral infections. Moreover, they represent an excellent approach for the discovery of new prophylactics and/or therapeutics for the treatment of poxvirus infections.IMPORTANCEDespite the eradication of variola virus (VARV), the causative agent of smallpox, poxviruses still represent an important threat to human health due to their possible use as bioterrorism agents and the emergence of zoonotic poxvirus diseases. Recombinant vaccinia viruses (rVV) expressing easily traceable fluorescent or luciferase reporter genes have significantly contributed to the progress of poxvirus research. However, rVV expressing one marker gene have several constraints for in vitro and in vivo studies, since both fluorescent and luciferase proteins impose certain limitations for specific applications. To overcome these limitations, we generated optimized rVV stably expressing both fluorescent (Scarlet or GFP) and luciferase (Nluc) reporter genes to easily track viral infection in vitro and in vivo. This new generation of double reporter-expressing rVV represent an excellent option to study viral infection dynamics in cultured cells and validated animal models of infection, and for the discovery of new poxvirus antiviral treatments.

Global deployment of an effective and safe vaccine is necessary to curtail the coronavirus disease 2019 (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Here, we evaluated a Newcastle disease virus (NDV)-based vectored-vaccine in mice and hamsters for its immunogenicity, safety and protective efficacy against SARS-CoV-2. Intranasal administration of recombinant (r)NDV-S vaccine expressing spike (S) protein of SARS-CoV-2 to mice induced high levels of SARS-CoV-2-specific neutralizing immunoglobulin A (IgA) and IgG2a antibodies and T cell-mediated immunity. Hamsters immunised with two doses of vaccine showed complete protection from lung infection, inflammation, and pathological lesions following SARS-CoV-2 challenge. Importantly, administration of two doses of intranasal rNDV-S vaccine significantly reduced the SARS-CoV-2 shedding in nasal turbinate and lungs in hamsters. Collectively, intranasal vaccination has the potential to control infection at the site of inoculation, which should prevent both clinical disease and virus transmission to halt the spread of the COVID-19 pandemic., Graphical Abstract

[Image: see text] A series of α-GalCer analogues containing an α-fluorocarbonyl moiety at the terminal position of the acyl chain were designed for targeting polar residues in the hydrophobic cavity of CD1d using a structure-based approach. The acyl chain length was efficiently adjusted by an asymmetric alkyne–alkyne cross coupling strategy, and the newly synthesized α-GalCer analogues showed the high Th2-selective activity of iNKT cells. The biased activity of ligands could be caused by the hydrogen-bonding interaction between ligands and CD1d according to the Th2-selective cytokine secretion and molecular docking studies.

An 18-year-old male Yorkshire Terrier was admitted with a history of neurological signs including dullness and progressive tetraparesis. Physical examination revealed bilaterally symmetrical alopecia and pot-bellied abdomen. Computed tomography and necropsy examination showed a mass across the frontal sinus and cerebral frontal lobe, bilateral adrenocortical hyperplasia, and hepatomegaly. Histopathologically, the tumor lesions consisted of sheets, nests, or cords of small- to medium-sized round-to-polyhedral cells. Adrenal cortex showed bilateral diffuse cellular proliferation, and some hepatocytes showed intracytoplasmic glycogen accumulation. Immunohistochemically, the tumor cells were positive for pancytokeratin, chromogranin-A, neuron-specific enolase, S100, synaptophysin, and thyroid transcription factor-1 but negative for microtubule-associated proein-2 and neurofilament, leading to the diagnosis of neuroendocrine tumor. These tumor cells were also positive for adrenocorticotropic hormone.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the viral pathogen responsible for the current coronavirus disease 2019 (COVID-19) pandemic. As of 19 May 2021, John Hopkins University’s COVID-19 tracking platform reported 3.3 million deaths associated with SARS-CoV-2 infection. Currently, the World Health Organization has granted emergency use listing (EUL) to six COVID-19 vaccine candidates. However, much of the pathogenesis observed during SARS-CoV-2 infection remains elusive. To gain insight into the contribution of individual accessory open reading frame (ORF) proteins in SARS-CoV-2 pathogenesis, we used our recently described reverse-genetics system approach to successfully engineer recombinant SARS-CoV-2 (rSARS-CoV-2) constructs; we removed individual viral ORF3a, −6, −7a, −7b, and −8 proteins from them, and we characterized the resulting recombinant viruses in vitro and in vivo. Our results indicate differences in plaque morphology, with ORF-deficient (ΔORF) viruses producing smaller plaques than those of the wild type (rSARS-CoV-2/WT). However, growth kinetics of ΔORF viruses were like those of rSARS-CoV-2/WT. Interestingly, infection of K18 human angiotensin-converting enzyme 2 (hACE2) transgenic mice with the ΔORF rSARS-CoV-2s identified ORF3a and ORF6 as the major contributors of viral pathogenesis, while ΔORF7a, ΔORF7b, and ΔORF8 rSARS-CoV-2s induced pathology comparable to that of rSARS-CoV-2/WT. This study demonstrates the robustness of our reverse-genetics system to generate rSARS-CoV-2 constructs and the major role for ORF3a and ORF6 in viral pathogenesis, providing important information for the generation of attenuated forms of SARS-CoV-2 for their implementation as live attenuated vaccines for the treatment of SARS-CoV-2 infection and associated COVID-19. IMPORTANCE Despite great efforts put forward worldwide to combat the current coronavirus disease 2019 (COVID-19) pandemic, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to be a human health and socioeconomic threat. Insights into the pathogenesis of SARS-CoV-2 and the contribution of viral proteins to disease outcome remain elusive. Our study aims (i) to determine the contribution of SARS-CoV-2 accessory open reading frame (ORF) proteins to viral pathogenesis and disease outcome and (ii) to develop a synergistic platform combining our robust reverse-genetics system to generate recombinant SARS-CoV-2 constructs with a validated rodent model of infection and disease. We demonstrate that SARS-CoV-2 ORF3a and ORF6 contribute to lung pathology and ultimately disease outcome in K18 hACE2 transgenic mice, while ORF7a, ORF7b, and ORF8 have little impact on disease outcome. Moreover, our combinatory platform serves as a foundation for generating attenuated forms of the virus to develop live attenuated vaccines for the treatment of SARS-CoV-2.

SUMMARYReplication-competent recombinant viruses expressing reporter genes provide valuable tools to investigate viral infection. Low levels of reporter gene expressed from previous reporter-expressing rSARS-CoV-2 have jeopardized their use to monitor the dynamics of SARS-CoV-2 infection in vitro or in vivo. Here, we report an alternative strategy where reporter genes were placed upstream of the viral nucleocapsid gene followed by a 2A cleavage peptide. The higher levels of reporter expression using this strategy resulted in efficient visualization of rSARS-CoV-2 in infected cultured cells and K18 hACE2 transgenic mice. Importantly, real-time viral infection was readily tracked using a non-invasive in vivo imaging system and allowed us to rapidly identify antibodies which are able to neutralize SARS-CoV-2 infection in vivo. Notably, these reporter-expressing rSARS-CoV-2 retained wild-type virus like pathogenicity in vivo, supporting their use to investigate viral infection, dissemination, pathogenesis and therapeutic interventions for the treatment of SARS-CoV-2 in vivo.

Summary/AbstractFACT (FAcilitates Chromatin Transcription) is a heterodimeric protein complex composed of SUPT16H and SSRP1, and a histone chaperone participating in chromatin remodeling during gene transcription. FACT complex is profoundly regulated, and contributes to both gene activation and suppression. Here we reported that SUPT16H, a subunit of FACT, is acetylated at lysine 674 (K674) of middle domain (MD), which involves TIP60 histone acetyltransferase. Such acetylation of SUPT16H is recognized by bromodomain protein BRD4, which promotes protein stability of SUPT16H. We further demonstrated that SUPT16H-BRD4 associates with histone modification enzymes (EZH2, HDAC1) and affects histone marks (H3K9me3, H3K27me3 and H3ac). BRD4 is known to profoundly regulate interferon (IFN) signaling, while such function of SUPT16H has never been explored. Surprisingly, our results revealed that SUPT16H genetic knockdown via RNAi or pharmacological inhibition by using its inhibitor, curaxin 137 (CBL0137), results in the induction of IFNs and interferon-stimulated genes (ISGs). Through this mechanism, CBL0137 is shown to efficiently inhibit infection of multiple viruses, including Zika, influenza, and SARS-CoV-2. Furthermore, we demonstrated that CBL0137 also causes the remarkable activation of IFN signaling in natural killer (NK) cells, which promotes the NK-mediated killing of virus-infected cells in a co-culture system using human primary NK cells. Overall, our studies unraveled the previously un-appreciated role of FACT complex in regulating IFN signaling in both epithelial and NK cells, and also proposed the novel application of CBL0137 to treat viral infections.

Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2) is the viral pathogen responsible for the current coronavirus disease 2019 (COVID-19) pandemic. To date, it is estimated that over 113 million individuals have been infected with SARS-CoV-2 and over 2.5 million human deaths have been recorded worldwide. Currently, three vaccines have been approved by the Food and Drug Administration for emergency use only. However much of the pathogenesis observed during SARS-CoV-2 infection remains elusive. To gain insight into the contribution of individual accessory open reading frame (ORF) proteins in SARS-CoV-2 pathogenesis, we used our recently described reverse genetics system approach to successfully engineer recombinant (r)SARS-CoV-2, where we individually removed viral 3a, 6, 7a, 7b, and 8 ORF proteins, and characterized these recombinant viruses in vitro and in vivo. Our results indicate differences in plaque morphology, with ORF deficient (ΔORF) viruses producing smaller plaques than those of the wild-type (rSARS-CoV-2/WT). However, growth kinetics of ΔORF viruses were like those of rSARS-CoV-2/WT. Interestingly, infection of K18 human angiotensin converting enzyme 2 (hACE2) transgenic mice with the ΔORF rSARS-CoV-2 identified ORF3a and ORF6 as the major contributors of viral pathogenesis, while ΔORF7a, ΔORF7b and ΔORF8 rSARS-CoV-2 induced comparable pathology to rSARS-CoV-2/WT. This study demonstrates the robustness of our reverse genetics system to generate rSARS-CoV-2 and the major role for ORF3a and ORF6 in viral pathogenesis, providing important information for the generation of attenuated forms of SARS-CoV-2 for their implementation as live-attenuated vaccines for the treatment of SARS-CoV-2 infection and associated COVID-19.IMPORTANCEDespite great efforts put forward worldwide to combat the current coronavirus disease 2019 (COVID-19) pandemic, Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2) continues to be a human health and socioeconomic threat. Insights into the pathogenesis of SARS-CoV-2 and contribution of viral proteins to disease outcome remains elusive. Our study aims to determine the contribution of SARS-CoV-2 accessory open reading frame (ORF) proteins in viral pathogenesis and disease outcome, and develop a synergistic platform combining our robust reverse genetics system to generate recombinant (r)SARS-CoV-2 with a validated rodent model of infection and disease. We demonstrated that SARS-CoV-2 ORF3a and ORF6 contribute to lung pathology and ultimately disease outcome in K18 hACE2 transgenic mice, while ORF7a, ORF7b, and ORF8 have little impact on disease outcome. Moreover, our combinatory platform serves as the foundation to generate attenuated forms of the virus to develop live-attenuated vaccines for the treatment of SARS-CoV-2.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the pathogen that causes coronavirus disease 2019 (COVID-19), has significantly impacted the human health and economic status worldwide. There is an urgent need to identify effective prophylactics and therapeutics for the treatment of SARS-CoV-2 infection and associated COVID-19., The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the pathogen responsible for coronavirus disease 2019 (COVID-19), has devastated public health services and economies worldwide. Despite global efforts to contain the COVID-19 pandemic, SARS-CoV-2 is now found in over 200 countries and has caused a death toll of over 1 million human lives as of November 2020. To date, only one Food and Drug Administration (FDA)-approved therapeutic drug (remdesivir) and a monoclonal antibody (MAb), bamlanivimab, are available for the treatment of SARS-CoV-2. As with other viruses, studying SARS-CoV-2 requires the use of secondary approaches to detect the presence of the virus in infected cells. To overcome this limitation, we have generated replication-competent recombinant SARS-CoV-2 (rSARS-CoV-2) constructs expressing fluorescent (Venus or mCherry) or bioluminescent (Nluc) reporter genes. Vero E6 cells infected with reporter-expressing rSARS-CoV-2 can be easily detected via fluorescence or luciferase expression and display a good correlation between reporter gene expression and viral replication. Moreover, rSARS-CoV-2 expressing reporter genes has plaque sizes and growth kinetics comparable to those of wild-type virus, rSARS-CoV-2/WT. We used these reporter-expressing rSARS-CoV-2 constructs to demonstrate their feasibility to identify neutralizing antibodies (NAbs) or antiviral drugs. Our results demonstrate that reporter-expressing rSARS-CoV-2 represents an excellent option to identify therapeutics for the treatment of SARS-CoV-2, where reporter gene expression can be used as a valid surrogate to track viral infection. Moreover, the ability to manipulate the viral genome opens the feasibility of generating viruses expressing foreign genes for their use as vaccines for the treatment of SARS-CoV-2 infection. IMPORTANCE Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the pathogen that causes coronavirus disease 2019 (COVID-19), has significantly impacted the human health and economic status worldwide. There is an urgent need to identify effective prophylactics and therapeutics for the treatment of SARS-CoV-2 infection and associated COVID-19. The use of fluorescent-protein- or luciferase-expressing reporter viruses has significantly advanced viral research. Here, we generated recombinant SARS-CoV-2 (rSARS-CoV-2) constructs expressing fluorescent (Venus and mCherry) or luciferase (Nluc) reporter genes and demonstrated that these viruses represent an excellent option to track viral infections in vitro. Importantly, reporter-expressing rSARS-CoV-2 constructs display growth kinetics and plaque phenotypes similar to those of their wild-type counterpart (rSARS-CoV-2/WT), demonstrating their usefulness for identifying drugs and/or neutralizing antibodies (NAbs) for the therapeutic treatment of SARS-CoV-2. Henceforth, these reporter-expressing rSARS-CoV-2 constructs can be used to interrogate large libraries of compounds and/or monoclonal antibodies (MAb), in high-throughput screening settings, to identify those with therapeutic potential against SARS-CoV-2.

An understanding of immunological mechanisms in kidney diseases has advanced using mouse kidneys. However, the profiling of immune cell subsets in human kidneys remains undetermined, particularly compared with mouse kidneys. Normal human kidneys were obtained from radically nephrectomised patients with urogenital malignancy (n = 15). Subsequently, human kidney immune cell subsets were analysed using multicolor flow cytometry and compared with subsets from C57BL/6 or BALB/c mice under specific pathogen-free conditions. Twenty kidney sections from healthy kidney donors or subjects without specific renal lesions were additionally analysed by immunohistochemistry. In human kidneys, 47% ± 12% (maximum 63%) of immune cells were CD3

Vaccine and antiviral development against SARS-CoV-2 infection or COVID-19 disease would benefit from validated small animal models. Here, we show that transgenic mice expressing human angiotensin-converting enzyme 2 (hACE2) by the human cytokeratin 18 promoter (K18 hACE2) represent a susceptible rodent model. K18 hACE2 transgenic mice succumbed to SARS-CoV-2 infection by day 6, with virus detected in lung airway epithelium and brain. K18 ACE2 transgenic mice produced a modest TH1/2/17 cytokine storm in the lung and spleen that peaked by day 2, and an extended chemokine storm that was detected in both lungs and brain. This chemokine storm was also detected in the brain at day 6. K18 hACE2 transgenic mice are, therefore, highly susceptible to SARS-CoV-2 infection and represent a suitable animal model for the study of viral pathogenesis, and for identification and characterization of vaccines (prophylactic) and antivirals (therapeutics) for SARS-CoV-2 infection and associated severe COVID-19 disease., Here, the authors characterize tissue-level SARS-CoV-2 infection and pathogenesis in transgenic mice expressing human angiotensin converting enzyme 2 (hACE2) by the human cytokeratin 18 promoter (K18-hACE2) and show that infection induces lethality, making the K18-hACE2 model suitable for vaccine and therapeutic evaluation.

Infectious coronavirus (CoV) disease 2019 (COVID-19) emerged in the city of Wuhan (China) in December 2019, causing a pandemic that has dramatically impacted public health and socioeconomic activities worldwide. A previously unknown coronavirus, severe acute respiratory syndrome CoV-2 (SARS-CoV-2), has been identified as the causative agent of COVID-19. To date, there are no U.S. Food and Drug Administration (FDA)-approved vaccines or therapeutics available for the prevention or treatment of SARS-CoV-2 infection and/or associated COVID-19 disease, which has triggered a large influx of scientific efforts to develop countermeasures to control SARS-CoV-2 spread. To contribute to these efforts, we have developed an infectious cDNA clone of the SARS-CoV-2 USA-WA1/2020 strain based on the use of a bacterial artificial chromosome (BAC). Recombinant SARS-CoV-2 (rSARS-CoV-2) was readily rescued by transfection of the BAC into Vero E6 cells. Importantly, BAC-derived rSARS-CoV-2 exhibited growth properties and plaque sizes in cultured cells comparable to those of the natural SARS-CoV-2 isolate. Likewise, rSARS-CoV-2 showed levels of replication similar to those of the natural isolate in nasal turbinates and lungs of infected golden Syrian hamsters. This is, to our knowledge, the first BAC-based reverse genetics system for the generation of infectious rSARS-CoV-2 that displays features in vivo similar to those of a natural viral isolate. This SARS-CoV-2 BAC-based reverse genetics will facilitate studies addressing several important questions in the biology of SARS-CoV-2, as well as the identification of antivirals and development of vaccines for the treatment of SARS-CoV-2 infection and associated COVID-19 disease.

An understanding of immunological mechanisms in kidney diseases has advanced using mouse kidneys. However, the profiling of immune cell subsets in human kidneys remains undetermined, particularly compared with mouse kidneys. Normal human kidneys were obtained from radically nephrectomised patients with urogenital malignancy (n = 15). Subsequently, human kidney immune cell subsets were analysed using multicolor flow cytometry and compared with subsets from C57BL/6 or BALB/c mice under specific pathogen-free conditions. Twenty kidney sections from healthy kidney donors or subjects without specific renal lesions were additionally analysed by immunohistochemistry. In human kidneys, 47% ± 12% (maximum 63%) of immune cells were CD3+ T cells. Kidney CD4+ and CD8+ T cells comprised 44% and 56% of total T cells. Of these, 47% ± 15% of T cells displayed an effector memory phenotype (CCR7− CD45RA− CD69−), and 48% ± 19% were kidney-resident cells (CCR7− CD45RA− CD69+). However, the proportions of human CD14+ and CD16+ myeloid cells were approximately 10% of total immune cells. A predominance of CD3+ T cells and a low proportion of CD14+ or CD68+ myeloid cells were also identified in healthy human kidney sections. In mouse kidneys, kidney-resident macrophages (CD11blow F4/80high) were the most predominant subset (up to 50%) but the proportion of CD3+ T cells was less than 20%. These results will be of use in studies in which mouse results are translated into human cases under homeostatic conditions or with disease.

© 2020 by the authors., Zika virus (ZIKV) was identified in 1947 in the Zika forest of Uganda and it has emerged recently as a global health threat, with recurring outbreaks and its associations with congenital microcephaly through maternal fetal transmission and Guillain-Barré syndrome. Currently, there are no United States (US) Food and Drug Administration (FDA)-approved vaccines or antivirals to treat ZIKV infections, which underscores an urgent medical need for the development of disease intervention strategies to treat ZIKV infection and associated disease. Drug repurposing offers various advantages over developing an entirely new drug by significantly reducing the timeline and resources required to advance a candidate antiviral into the clinic. Screening the ReFRAME library, we identified ten compounds with antiviral activity against the prototypic mammarenavirus lymphocytic choriomeningitis virus (LCMV). Moreover, we showed the ability of these ten compounds to inhibit influenza A and B virus infections, supporting their broad-spectrum antiviral activity. In this study, we further evaluated the broad-spectrum antiviral activity of the ten identified compounds by testing their activity against ZIKV. Among the ten compounds, Azaribine (SI-MTT = 146.29), AVN-944 (SI-MTT = 278.16), and Brequinar (SI-MTT = 157.42) showed potent anti-ZIKV activity in post-treatment therapeutic conditions. We also observed potent anti-ZIKV activity for Mycophenolate mofetil (SI-MTT = 20.51), Mycophenolic acid (SI-MTT = 36.33), and AVN-944 (SI-MTT = 24.51) in pre-treatment prophylactic conditions and potent co-treatment inhibitory activity for Obatoclax (SI-MTT = 60.58), Azaribine (SI-MTT = 91.51), and Mycophenolate mofetil (SI-MTT = 73.26) in co-treatment conditions. Importantly, the inhibitory effect of these compounds was strain independent, as they similarly inhibited ZIKV strains from both African and Asian/American lineages. Our results support the broad-spectrum antiviral activity of these ten compounds and suggest their use for the development of antiviral treatment options of ZIKV infection., This research was funded by the National Institute of Health (NIH) grant number 1R21AI120500 to FA and L.M.-S.

Various microorganisms are involved in nitrogen removal, and their group compositions depend closely on operating parameters. The structures and functions of nitrification microorganisms in full-scale anaerobic-anoxic–oxic (A2/O) and oxidation ditch processes were analyzed using metagenomics and canonical correspondence analysis. The community structure of ammonia-oxidizing archaea in the oxidation ditch was 3.8 (winter) – 6.3 (summer) times higher than in A2/O, and the complete ammonia oxidizer was only found in the oxidation ditch process. The canonical correspondence analysis of various environmental variables showed that Nitrosomonadales, Crenarchaeota, and Nitrospira inopinata correlate highly with nitrification, and Nitrospira was involved in NO2−-N oxidation rather than Nitrobacter. The longer solid and hydraulic retention times in the oxidation ditch were more effective in achieving a wider range of novel nitrification than A2/O. This result indicates that microbial communities of novel nitrifiers and ammonia-oxidizing archaea improved in the oxidation ditch process, significantly contributing to stable nitrogen removal.

Towards the end of 2019, a novel coronavirus (CoV) named severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), genetically similar to severe acute respiratory syndrome coronavirus-1 (SARS-CoV-1), emerged in Wuhan, Hubei province of China, and has been responsible of coronavirus disease 2019 (COVID-19) in humans. Since its first report, SARS-CoV-2 has resulted in a global pandemic, with over 10 million human infections and over 560,000 deaths reported worldwide at the end of June 2020. Currently, there are no United States (US) Food and Drug Administration (FDA)-approved vaccines and/or antivirals licensed against SARS-CoV-2, and the high economical and health impact of SARS-CoV-2 has placed global pressure on the scientific community to identify effective prophylactic and therapeutic treatments for the treatment of SARS-CoV-2 infection and associated COVID-19 disease. While some compounds have been already reported to reduce SARS-CoV-2 infection and a handful of monoclonal antibodies (mAbs) have been described that neutralize SARS-CoV-2, there is an urgent need for the development and standardization of assays which can be used in high through-put screening (HTS) settings to identify new antivirals and/or neutralizing mAbs against SARS-CoV-2. Here, we described a rapid, accurate and highly reproducible plaque reduction microneutralization (PRMNT) assay that can be quickly adapted for the identification and characterization of both neutralizing mAbs and antivirals against SARS-CoV-2. Importantly, our MNA is compatible with HTS settings to interrogate large and/or complex libraries of mAbs and/or antivirals to identify those with neutralizing and/or antiviral activity, respectively, against SARS-CoV-2.

Vaccine and antiviral development against SARS-CoV-2 infection or COVID-19 disease currently lacks a validated small animal model. Here, we show that transgenic mice expressing human angiotensin converting enzyme 2 (hACE2) by the human cytokeratin 18 promoter (K18 hACE2) represent a susceptible rodent model. K18 hACE2-transgenic mice succumbed to SARS-CoV-2 infection by day 6, with virus detected in lung airway epithelium and brain. K18 ACE2-transgenic mice produced a modest TH1/2/17 cytokine storm in the lung and spleen that peaked by day 2, and an extended chemokine storm that was detected in both lungs and brain. This chemokine storm was also detected in the brain at day 4. K18 hACE2-transgenic mice are, therefore, highly susceptible to SARS-CoV-2 infection and represent a suitable animal model for the study of viral pathogenesis, and for identification and characterization of vaccines (prophylactic) and antivirals (therapeutics) for SARS-CoV-2 infection and associated severe COVID-19 disease.

Although seasonal influenza vaccines block most predominant influenza types and subtypes, humans still remain vulnerable to waves of seasonal and new potential pandemic influenza viruses for which no immunity may exist because of viral antigenic drift and/or shift, respectively. Previously, we have described a human monoclonal antibody (hMAb), KPF1, which was produced in human embryonic kidney 293T cells (KPF1-HEK) with broad and potent neutralizing activity against H1N1 influenza A viruses (IAV)in vitro, and prophylactic and therapeutic activitiesin vivo. In this study, we produced hMAb KPF1 in tobacco plants (KPF1-Antx) and demonstrate how the plant-produced KPF1-Antx hMAb possesses similar biological activity compared with the mammalian produced KPF1-HEK hMAb. KPF1-Antx hMAb shows broad binding to recombinant HA proteins and H1N1 IAV, including A/California/04/2009 (pH1N1)in vitro, that are comparable to those observed with KPF1-HEK hMAb. Importantly, prophylactic administration of KPF1-Antx hMAb to guinea pigs prevented pH1N1 infection and transmission in both prophylactic and therapeutic experiments, substantiating its clinical potential to prevent and treat H1N1 infections. Collectively, this study demonstrates, for the first time, that plant-produced influenza hMAbs have similarin vitroandin vivobiological properties to those produced in mammalian cells. Because of the many advantages of plant-produced hMAbs, such as rapid batch production, low cost, and the absence of mammalian cell products, they represent an alternative strategy for the production of immunotherapeutics for the treatment of influenza viral infections, including emerging seasonal and/or pandemic strains.

© The Author(s) 2020., Zika virus (ZIKV) is a mosquito-borne member of the Flaviviridae family that has been known to circulate for decades causing mild febrile illness. The more recent ZIKV outbreaks in the Americas and the Caribbean associated with congenital malformations and Guillain-Barré syndrome in adults have placed public health officials in high alert and highlight the significant impact of ZIKV on human health. New technologies to study the biology of ZIKV and to develop more effective prevention options are highly desired. In this study we demonstrate that direct delivery in mice of an infectious ZIKV cDNA clone allows the rescue of recombinant (r)ZIKV in vivo. A bacterial artificial chromosome containing the sequence of ZIKV strain Paraiba/2015 under the control of the cytomegalovirus promoter was complexed with a commercial transfection reagent and administrated using different routes in type-I interferon receptor deficient A129 mice. Clinical signs and death associated with ZIKV viremia were observed in mice. The rZIKV recovered from these mice remained fully virulent in a second passage in mice. Interestingly, infectious rZIKV was also recovered after intraperitoneal inoculation of the rZIKV cDNA in the absence of transfection reagent. Further expanding these studies, we demonstrate that a single intraperitoneal inoculation of a cDNA clone encoding an attenuated rZIKV was safe, highly immunogenic, and provided full protection against lethal ZIKV challenge. This novel in vivo reverse genetics method is a potentially suitable delivery platform for the study of wild-type and live-attenuated ZIKV devoid of confounding factors typical associated with in vitro systems. Moreover, our results open the possibility of employing similar in vivo reverse genetic approaches for the generation of other viruses and, therefore, change the way we will use reverse genetics in the future., This research was funded by the Spanish Ministry of Economy and Competitiveness (MINECO) (grant number BFU2016-79127-R) to F.A. and the National Institute of Health (NIH) (grant number 1R21AI120500) to F.A. and L.M.-S. D.R.P. is partially supported the Georgia Research Alliance, the Georgia Poultry Federation and by a subcontract from the Center for Research on Infuenza Pathogenesis (CRIP) under contract HHSN272201400008C from the National Institute of Allergy and Infectious Diseases (NIAID) Centers for Infuenza Research and Surveillance (CEIRS).

Microbial electrolysis cells (MECs) are being studied to improve the efficiency of anaerobic digesters and biogas production. In the present study, we investigated the effects of electrochemical reactions in AD-MEC (anaerobic digester combined with MECs) on changes in the microbial communities of bulk sludge through 454-pyrosequencing analysis, as well as the effect of these changes on anaerobic digestion. Methanobacterium beijingense and Methanobacterium petrolearium were the dominant archaeal species in AD, while Methanosarcina thermophila and Methanobacterium formicicum were dominant in AD-MEC at steady-state. There were no substantial differences in dominant bacterial species. Clostridia class was more abundant than Bacteroidia class in both reactors. Compared to AD, AD-MEC showed a 40% increase in overall bacterial population, increasing the removal of organic matters and the conversion of volatile fatty acids (VFAs). Thus, the MEC reaction more effectively converts organic matters to VFAs and activates microbial communities favorable for methane production.

Influenza A (IAV) and influenza B (IBV) viruses are highly contagious pathogens that cause fatal respiratory disease every year, with high economic impact. In addition, IAV can cause pandemic infections with great consequences when new viruses are introduced into humans. In this study, we evaluated 10 previously described compounds with antiviral activity against mammarenaviruses for their ability to inhibit IAV infection using our recently described bireporter influenza A/Puerto Rico/8/34 (PR8) H1N1 (BIRFLU). Among the 10 tested compounds, eight (antimycin A [AmA], brequinar [BRQ], 6-azauridine, azaribine, pyrazofurin [PF], AVN-944, mycophenolate mofetil [MMF], and mycophenolic acid [MPA]), but not obatoclax or Osu-03012, showed potent anti-influenza virus activity under posttreatment conditions [median 50% effective concentration (EC(50)) = 3.80 nM to 1.73 μM; selective index SI for 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay, >28.90 to 13,157.89]. AmA, 6-azauridine, azaribine, and PF also showed potent inhibitory effect in pretreatment (EC(50) = 0.14 μM to 0.55 μM; SI-MTT = 70.12 to >357.14) or cotreatment (EC(50) = 34.69 nM to 7.52 μM; SI-MTT = 5.24 to > 1,441.33) settings. All of the compounds tested inhibited viral genome replication and gene transcription, and none of them affected host cellular RNA polymerase II activities. The antiviral activity of the eight identified compounds against BIRFLU was further confirmed with seasonal IAVs (A/California/04/2009 H1N1 and A/Wyoming/3/2003 H3N2) and an IBV (B/Brisbane/60/2008, Victoria lineage), demonstrating their broad-spectrum prophylactic and therapeutic activity against currently circulating influenza viruses in humans. Together, our results identified a new set of antiviral compounds for the potential treatment of influenza viral infections. IMPORTANCE Influenza viruses are highly contagious pathogens and are a major threat to human health. Vaccination remains the most effective tool to protect humans against influenza infection. However, vaccination does not always guarantee complete protection against drifted or, more noticeably, shifted influenza viruses. Although U.S. Food and Drug Administration (FDA) drugs are approved for the treatment of influenza infections, influenza viruses resistant to current FDA antivirals have been reported and continue to emerge. Therefore, there is an urgent need to find novel antivirals for the treatment of influenza viral infections in humans, a search that could be expedited by repurposing currently approved drugs. In this study, we assessed the influenza antiviral activity of 10 compounds previously shown to inhibit mammarenavirus infection. Among them, eight drugs showed antiviral activities, providing a new battery of drugs that could be used for the treatment of influenza infections.

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. Electronic supplementary material The online version of this article (10.1186/s13567-018-0619-6) contains supplementary material, which is available to authorized users.

Bacterial communities change in bulk solution of anaerobic digestion (AD) and bio-electrochemical anaerobic digestion reactors (BEAD) were monitored at each organic loading rate (OLR) to investigate the effect of voltage supply on bacterial species change in bulk solution. Chemical oxygen demand (COD) degradation and methane production from AD and BEAD reactors were also analyzed by gradually increasing food waste OLR. The BEAD reactor maintained stable COD removal and methane production at 6.0 kg/m3·d. The maximum OLR of AD reactor for optimal operation was 4.0 kg/m3·d. pH and alkalinity decline and volatile fatty acid (VFA) accumulation, which are the problem in high load anaerobic digestion of readily decomposable food wastes, were again the major factors destroying the optimal operation condition of the AD reactor at 6.0 kg/m3·d. Contrarily, the electrochemically activated dense communities of exoelectrogenic bacteria and VFA-oxidizing bacteria prevented VFAs from accumulating inside the BEAD reactor. This maintained stable pH and alkalinity conditions, ultimately contributing to stable methane production.

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., 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 Sambucus nigra lectin, but not by the α2,3-linked specific sialidase or by Maackia amurensis 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. IMPORTANCE 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.

© 2019 Park, Ávila-Pérez, Madere, Hilimire, Nogales, Almazán and Martínez-Sobrido., Zika virus (ZIKV) is one of the recently emerging vector-borne viruses in humans and is responsible for severe congenital abnormalities such as microcephaly in the Western Hemisphere. Currently, only a few vaccine candidates and therapeutic drugs are being developed for the treatment of ZIKV infections, and as of yet none are commercially available. The polyanionic aromatic compound aurintricarboxylic acid (ATA) has been shown to have a broad-spectrum antimicrobial and antiviral activity. In this study, we evaluated ATA as a potential antiviral drug against ZIKV replication. The antiviral activity of ATA against ZIKV replication in vitro showed median inhibitory concentrations (IC50) of 13.87 ± 1.09 μM and 33.33 ± 1.13 μM in Vero and A549 cells, respectively; without showing any cytotoxic effect in both cell lines (median cytotoxic concentration (CC50) > 1,000 μM). Moreover, ATA protected both cell types from ZIKV-induced cytopathic effect (CPE) and apoptosis in a time- and concentration-dependent manner. In addition, pre-treatment of Vero cells with ATA for up to 72 h also resulted in effective suppression of ZIKV replication with similar IC50. Importantly, the inhibitory effect of ATA on ZIKV infection was effective against strains of the African and Asian/American lineages, indicating that this inhibitory effect was not strain dependent. Overall, these results demonstrate that ATA has potent inhibitory activity against ZIKV replication and may be considered as a potential anti-ZIKV therapy for future clinical evaluation., This work was supported in part by the National Institutes of Health (NIH) grant 1R21AI130500 to LM-S and FA.

© 2019 Journal of Visualized Experiments., The association of Zika virus (ZIKV) infection with neurological complications during the recent worldwide outbreak and the lack of approved vaccines and/or antivirals have underscored the urgent need to develop ZIKV reverse genetic systems to facilitate the study of ZIKV biology and the development of therapeutic and/or prophylactic approaches. However, like with other flaviviruses, the generation of ZIKV full-length infectious cDNA clones has been hampered due to the toxicity of viral sequences during its amplification in bacteria. To overcome this problem, we have developed a nontraditional approach based on the use of bacterial artificial chromosomes (BACs). Using this approach, the full-length cDNA copy of the ZIKV strain Rio Grande do Norte Natal (ZIKV-RGN) is generated from four synthetic DNA fragments and assembled into the single-copy pBeloBAC11 plasmid under the control of the human cytomegalovirus (CMV) immediate-early promoter. The assembled BAC cDNA clone is stable during propagation in bacteria, and infectious recombinant (r)ZIKV is recovered in Vero cells after transfection of the BAC cDNA clone. The protocol described here provides a powerful technique for the generation of infectious clones of flaviviruses, including ZIKV, and other positive-strand RNA viruses, particularly those with large genomes that have stability problems during bacterial propagation., This work was supported in part by grants from the Spanish Ministry of Economy and Competitiveness (MINECO, grant number BFU2016-79127-R) to F.A.T. and the National Institutes of Health (NIH, grant number 1R21AI120500) to L.M.S. and F.A.T.

Highlights • We have developed an experimental approach for the selection, identification, and characterization of SARS-CoV-2 MARMs. • This assay can be used to identify AA residues in the S protein required for mNAb-mediated inhibition of viral infection. • Our assay can be used to predict potential natural drift variants that could emerge upon implementation of therapeutic mNAbs. • We also describe experimental approaches to evaluate MARM viral fitness and how to compare them to WT SARS-CoV-2., The use of monoclonal neutralizing antibodies (mNAbs) is being actively pursued as a viable intervention for the treatment of Severe Acute Respiratory Syndrome CoV-2 (SARS-CoV-2) infection and associated coronavirus disease 2019 (COVID-19). While highly potent mNAbs have great therapeutic potential, the ability of the virus to mutate and escape recognition and neutralization of mNAbs represents a potential problem in their use for the therapeutic management of SARS-CoV-2. Studies investigating natural or mNAb-induced antigenic variability in the receptor binding domain (RBD) of SARS-CoV-2 Spike (S) glycoprotein, and their effects on viral fitness are still rudimentary. In this manuscript we described experimental approaches for the selection, identification, and characterization of SARS-CoV-2 monoclonal antibody resistant mutants (MARMs) in cultured cells. The ability to study SARS-CoV-2 antigenic drift under selective immune pressure by mNAbs is important for the optimal implementation of mNAbs for the therapeutic management of COVID-19. This will help to identify essential amino acid residues in the viral S glycoprotein required for mNAb-mediated inhibition of viral infection, to predict potential natural drift variants that could emerge upon implementation of therapeutic mNAbs, as well as vaccine prophylactic treatments for SARS-CoV-2 infection. Additionally, it will also enable the assessment of MARM viral fitness and its potential to induce severe infection and associated COVID-19 disease.

This study attempted to enhance sulfidogenic activity via sulfate-reducing bacteria (SRB) enrichment and minimize organic carbon loss by methanogen inhibition in the sulfidogenic stage of a two-stage anaerobic digestion system (TSADS). To enrich SRB in the sulfidogenic stage, batch tests were performed with various granular sludge pretreatments. Starvation was the most effective pretreatment, increasing SO42− removal and minimizing chemical oxygen demand (COD) loss by inhibiting methanogen activity. Microbial community analysis showed that Desulfovibrio, Desulfotomaculum, and Syntrophobacter were the dominant SRB in the sulfidogenic stage (5.0%, 3.1%, and 2.4%, respectively). This enabled SO42− reduction (86%) and volatile fatty acid production (55% of fed COD) at a hydraulic retention time (HRT) of 4 h. Conversely, biogas with a reduced H2S content (110 ppmv) was produced in the methanogenic stage (HRT = 6 h). A granular sludge comparison revealed differences in their ecology, structure, and extracellular polymeric substance characteristics. Economic feasibility analysis demonstrated that TSADS can lead to a cost reduction of $80–90/1,000 m3 CH4 compared to single-stage anaerobic digestion.

Highlights • We have developed a rapid, accurate, and highly reproducible plaque reduction microneutralization (PRMNT) assay for SARS-CoV-2. • Our PRMNT assay allows to identify and characterize antibodies with neutralizing activity against SARS-CoV-2. • Likewise, our PRMNT assay can be used to find compounds with antiviral activity against SARS-CoV-2. • The PRMNT assay can be developed using peroxidase or infrared staining readouts. • Our PRMNT assay can be adapted to HTS settings to interrogate complex library of SARS-CoV-2 inhibitors., Towards the end of 2019, a novel coronavirus (CoV) named severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), genetically similar to severe acute respiratory syndrome coronavirus (SARS-CoV), emerged in Wuhan, Hubei province of China, and has been responsible for coronavirus disease 2019 (COVID-19) in humans. Since its first report, SARS-CoV-2 has resulted in a global pandemic, with over 10 million human infections and over 560,000 deaths reported worldwide at the end of June 2020. Currently, there are no United States (US) Food and Drug Administration (FDA)-approved vaccines and/or antivirals licensed against SARS-CoV-2. The high economical and health impacts of SARS-CoV-2 has placed global pressure on the scientific community to identify effective prophylactic and therapeutic treatments for SARS-CoV-2 infection and associated COVID-19 disease. While some compounds have been already reported to reduce SARS-CoV-2 infection and a handful of monoclonal antibodies (mAbs) have been described that neutralize SARS-CoV-2, there is an urgent need for the development and standardization of assays which can be used in high through-put screening (HTS) settings to identify new antivirals and/or neutralizing mAbs against SARS-CoV-2. Here, we described a rapid, accurate, and highly reproducible plaque reduction microneutralization (PRMNT) assay that can be quickly adapted for the identification and characterization of both neutralizing mAbs and antivirals against SARS-CoV-2. Importantly, our MNA is compatible with HTS settings to interrogate large and/or complex libraries of mAbs and/or antivirals to identify those with neutralizing and/or antiviral activity, respectively, against SARS-CoV-2.

This study analyzed eleven genomic segments of three Korean porcine G8P[7] group A rotavirus (RVA) strains. Phylogenetically, these strains contained two bovine-like and nine porcine-like genomic segments. Eight genes (VP1, VP2, VP6 and NSP1-NSP5) of strains 156-1 and 42-1 and seven genes (VP1, VP2, VP6 and NSP2-NSP5) of strain C-1 clustered closely with porcine and porcine-like animal strains and distantly from typical human Wa-like strains. The VP3-M2 genotype of these strains clustered closely with bovine-like strains, but distantly with typical human DS-1-like strains. These data indicate that multiple reassortments involving porcine and bovine RVA strains in Korea must have occurred. ispartof: Archives of Virology vol:161 issue:10 pages:2835-2841 ispartof: location:Austria status: published

International audience; AbstractCaliciviruses 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.

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., The genus Sapovirus belongs to the family Caliciviridae, 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. IMPORTANCE 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.

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., 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. IMPORTANCE 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.

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.

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.

Some viruses within the Caliciviridae 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 y 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. IMPORTANCE 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.

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.

Although reassortment is one of the most important characteristics of group A rotavirus (RVA) evolution, the host range restriction and/or virulence of reassortant RVAs remain largely unknown. The porcine 174-1 strain isolated from a diarrheic piglet was identified as a reassortant strain, harboring the same genotype constellation as the previously characterized bovine strain KJ56-1. Owing to its same genotype constellation, the pathogenicity of the porcine strain 174-1 in piglets and calves was examined for comparison with that of the bovine reassortant KJ56-1 strain, whose pathogenicity has already been demonstrated in piglets and calves. The porcine 174-1 strain induced diarrhea and histopathological changes in the small intestine of piglets and calves, whereas KJ56-1 had been reported to be virulent only in piglets, but not in calves. Therefore, full genomic sequences of 174-1 and KJ56-1 strains were analyzed to determine whether specific mutations might be associated with clinical and pathological phenotypes. Sequence alignment between the 174-1 and KJ56-1 strains detected one nucleotide substitution at the 3' untranslated region of the NSP3 gene and 16 amino acid substitutions at the VP7, VP4, VP1, VP3, NSP1 and NSP4 genes. These mutations may be critical molecular determinants for different virulence and/or pathogenicity of each strain. This study presents new insights into the host range restriction and/or virulence of RVAs.

To evaluate the prevalence and genetic diversity of porcine sapeloviruses (PSVs) in Korea, a total of 100 diarrhea fecal samples from pigs were analyzed by RT-PCR and nested PCR assays with primer pairs specific for the VP1 gene. Overall, 34 % of the diarrhea samples tested positive for PSV, and a high proportion of infections occurred along with a variety of other enteric viruses and bacteria. Genomic and phylogenetic analysis of the VP1 genes revealed pronounced genetic diversities between PSVs from Korean and elsewhere. Our results indicate that PSV infections are very common in Korean pigs with diarrhea. The infecting strains are genetically diverse. Electronic supplementary material The online version of this article (doi:10.1007/s00705-013-1901-6) contains supplementary material, which is available to authorized users.

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.

Group A rotaviruses (RVAs) are agents causing severe gastroenteritis in infants and young animals. G9 RVA strains are believed to have originated from pigs. However, this genotype has emerged as the fifth major human RVA genotype worldwide. To better understand the relationship between human and porcine RVA strains, complete RVA genome data are needed. For human RVA strains, the number of complete genome data have grown exponentially. However, there is still a lack of complete genome data on porcine RVA strains. Recently, G9 RVA strains have been identified as the third most important genotype in diarrheic pigs in South Korea in combinations with P[7] and P[23]. This study is the first report on complete genome analyses of 1 G9P[7] and 3 G9P[23] porcine RVA strains, resulting in the following genotype constellation: G9-P[7]/P[23]-I5-R1-C1-M1-A8-N1-T1-E1-H1. By comparisons of these genotype constellations, it was revealed that the Korean G9P[7] and G9P[23] RVA strains possessed a typical porcine RVA backbone, similar to other known porcine RVA strains. However, detailed phylogenetic analyses revealed the presence of intra-genotype reassortments among porcine RVA strains in South Korea. Thus, our data provide genetic information of G9 RVA strains increasingly detected in both humans and pigs, and will help to establish the role of pigs as a source or reservoir for novel human RVA strains.

Orthoreoviruses infect virtually all mammalian species, causing systemic infections including mild gastrointestinal and respiratory illnesses. However, little is known about the prevalence or genetic diversity of porcine orthoreoviruses in South Korea. We examined 237 diarrheic fecal samples collected from 78 pig farms around the country. RT-PCR utilizing primers specific for the L1 gene of mammalian orthoreoviruses showed that 45 (19.0%) samples were positive. The 10 strains isolated from orthoreovirus-positive samples formed typical perinuclear cytoplasmic inclusion bodies and had an atypical hemagglutination pattern; these are characteristics of type 3 orthoreovirus. Phylogenetic analysis of the S1 gene in these 10 Korean and other strains showed that type 3 orthoreoviruses could be divided into four lineages; the 10 Korean strains were included in porcine lineage IV, along with T3/porcine/Sichuan/2006. Sequence analysis showed that strains in lineage IV had nucleotide identities of 97.0–98.1% and deduced amino acid identities of 96.4–98.2%. Sequence analysis of the σ1 protein, a viral attachment protein, revealed that the amino acid sequences associated with neurotropism (amino acids 198–204, 249I, 350D, and 419E) were highly conserved among the Korean strains, confirming that neural tropism was present. In conclusion, our findings suggest that porcine orthoreovirus infections are endemic in pig farms in South Korea and that the 10 novel Korean porcine orthoreoviruses belong to porcine lineage IV of type 3 orthoreovirus. In addition, sequence analysis of S1 genes encoding the σ1 protein showed that the 9 of 10 Korean porcine orthoreoviruses exhibited neural tropism.

The present study investigated the effects of azuki bean (Vigna angularis) extract (VAE) on the progress of atopic dermatitis (AD)-like skin lesions in NC/Nga mice induced by 1-chloro-2,4-dinitrobenzene. The efficacy of VAE in NC/Nga mice was determined by measuring gross and histological skin lesions, serum IgE levels, eosinophil ratio in peripheral leucocytes, and mRNA expression levels of interleukin (IL)-4, tumour necrosis factor (TNF)-α and interferon (IFN)-γ in splenocytes. Continuous ingestion of VAE inhibited the development of the AD-like skin lesions in a dose-dependent manner. In the VAE-treated mice, the numbers of mast cells in the skin, eosinophil ratio in peripheral leucocytes, relative mRNA expression of inflammatory cytokines in the spleen, and serum IgE levels were significantly reduced. Results suggest that VAE can inhibit the development of AD-like skin lesions in NC/Nga mice by regulating immune mediators and cells, and may be an effective alternative therapy for AD.