A novel optimized pre-embedding antibody-labelling correlative light electron microscopy technique.

In the intricate environment of a cell, many studies seek to discover the location of specific events or objects of interest. Advances in microscopy in recent years have allowed for high detail views of specific areas of cells of interest using correlative light electron microscopy (CLEM). While this powerful technique allows for the correlation of a specific area of fluorescence on a confocal microscope with that same area in an electron microscope, it is most often used to study tagged proteins of interest. This method adapts the correlative method for use with antibody labelling. We have shown that some cellular structures are more sensitive than others to this process and that this can be a useful technique for laboratories where tagged proteins or viruses, or dedicated CLEM instruments are not available.

Preparation of Immunofluorescently Labeled Tissue Sections for Imaging at Low and High Magnifications in the Confocal Microscope.

The confocal laser scanning microscope allows us to examine tissue sections in greater detail than a widefield fluorescence microscope. However, this requires samples to be better preserved than standard cryostat sections, which are not usually aldehyde-fixed. Thick sections (approximately 70 µm) of formaldehyde-fixed tissue can be cut using a vibrating microtome and subsequently labeled with primary and secondary fluorescent antibodies and/or fluorescent stains. When imaged in the confocal microscope, these samples allow us to collect high-resolution images, detailing the intracellular location of multiple proteins and structures. In this chapter, we describe the technique used to prepare vibrating microtome sections, using porcine tissue infected with African swine fever virus as an example. This technique can easily be applied to any animal tissue with any suitable combination of antibodies, depending on the hypothesis.

Birnaviridae Virus Factories Show Features of Liquid-Liquid Phase Separation and Are Distinct from Paracrystalline Arrays of Virions Observed by Electron Microscopy.

To gain more information about the nature of Birnaviridae virus factories (VFs), we used a recombinant infectious bursal disease virus (IBDV) expressing split-GFP11 tagged to the polymerase (VP1) that we have previously shown is a marker for VFs in infected cells expressing GFP1-10. We found that VFs colocalized with 5-ethynyl uridine in the presence of actinomycin, demonstrating they contained newly synthesized viral RNA, and VFs were visible in infected cells that were fixed and permeabilized with digitonin, demonstrating that they were not membrane bound. Fluorescence recovery after photobleaching (FRAP) a region of interest within the VFs occurred rapidly, recovering from approximately 25% to 87% the original intensity over 146 s, and VFs were dissolved by 1,6-hexanediol treatment, demonstrating they showed properties consistent with liquid-liquid phase separation. There was a lower colocalization of the VF GFP signal with the capsid protein VP2 (Manders' coefficient [MC] 0.6), compared to VP3 (MC, 0.9), which prompted us to investigate the VF ultrastructure by transmission electron microscopy (TEM). In infected cells, paracrystalline arrays (PAs) of virions were observed in the cytoplasm, as well as discrete electron dense regions. Using correlative light and electron microscopy (CLEM), we observed that the electron dense regions correlated with the GFP signal of the VFs, which were distinct from the PAs. In summary, Birnaviridae VFs contain newly synthesized viral RNA, are not bound by a membrane, show properties consistent with liquid-liquid phase separation, and are distinct from the PAs observed by TEM. IMPORTANCE Members of the Birnaviridae infect birds, fish and insects, and are responsible for diseases of significant economic importance to the poultry industry and aquaculture. Despite their importance, how they replicate in cells remains poorly understood. Here, we show that the Birnaviridae virus factories are not membrane bound, demonstrate properties consistent with liquid-liquid phase separation, and are distinct from the paracrystalline arrays of virions observed by transmission electron microscopy, enhancing our fundamental knowledge of virus replication that could be used to develop strategies to control disease, or optimize their therapeutic application.

Coronavirus RNA Synthesis Takes Place within Membrane-Bound Sites.

Infectious bronchitis virus (IBV), a gammacoronavirus, is an economically important virus to the poultry industry, as well as a significant welfare issue for chickens. As for all positive strand RNA viruses, IBV infection causes rearrangements of the host cell intracellular membranes to form replication organelles. Replication organelle formation is a highly conserved and vital step in the viral life cycle. Here, we investigate the localization of viral RNA synthesis and the link with replication organelles in host cells. We have shown that sites of viral RNA synthesis and virus-related dsRNA are associated with one another and, significantly, that they are located within a membrane-bound compartment within the cell. We have also shown that some viral RNA produced early in infection remains within these membranes throughout infection, while a proportion is trafficked to the cytoplasm. Importantly, we demonstrate conservation across all four coronavirus genera, including SARS-CoV-2. Understanding more about the replication of these viruses is imperative in order to effectively find ways to control them.

Generation of Antibodies against Foot-and-Mouth-Disease Virus Capsid Protein VP4 Using Hepatitis B Core VLPs as a Scaffold.

The picornavirus foot-and-mouth disease virus (FMDV) is the causative agent of the economically important disease of livestock, foot-and-mouth disease (FMD). VP4 is a highly conserved capsid protein, which is important during virus entry. Previous published work has shown that antibodies targeting the N-terminus of VP4 of the picornavirus human rhinovirus are broadly neutralising. In addition, previous studies showed that immunisation with the N-terminal 20 amino acids of enterovirus A71 VP4 displayed on the hepatitis B core (HBc) virus-like particles (VLP) can induce cross-genotype neutralisation. To investigate if a similar neutralising response against FMDV VP4 could be generated, HBc VLPs displaying the N-terminus of FMDV VP4 were designed. The N-terminal 15 amino acids of FMDV VP4 was inserted into the major immunodominant region. HBc VLPs were also decorated with peptides of the N-terminus of FMDV VP4 attached using a HBc-spike binding tag. Both types of VLPs were used to immunise mice and the resulting serum was investigated for VP4-specific antibodies. The VLP with VP4 inserted into the spike, induced VP4-specific antibodies, however the VLPs with peptides attached to the spikes did not. The VP4-specific antibodies could recognise native FMDV, but virus neutralisation was not demonstrated. This work shows that the HBc VLP presents a useful tool for the presentation of FMDV capsid epitopes.

Quantifying and Modeling the Acquisition and Retention of Lumpy Skin Disease Virus by Hematophagus Insects Reveals Clinically but Not Subclinically Affected Cattle Are Promoters of Viral Transmission and Key Targets for Control of Disease Outbreaks.

Lumpy skin disease virus (LSDV) is a vector-transmitted poxvirus that causes disease in cattle. Vector species involved in LSDV transmission and their ability to acquire and transmit the virus are poorly characterized. Using a highly representative bovine experimental model of lumpy skin disease, we fed four model vector species (Aedes aegypti, Culex quinquefasciatus, Stomoxys calcitrans, and Culicoides nubeculosus) on LSDV-inoculated cattle in order to examine their acquisition and retention of LSDV. Subclinical disease was a more common outcome than clinical disease in the inoculated cattle. Importantly, the probability of vectors acquiring LSDV from a subclinical animal (0.006) was very low compared with that from a clinical animal (0.23), meaning an insect feeding on a subclinical animal was 97% less likely to acquire LSDV than one feeding on a clinical animal. All four potential vector species studied acquired LSDV from the host at a similar rate, but Aedes aegypti and Stomoxys calcitrans retained the virus for a longer time, up to 8 days. There was no evidence of virus replication in the vector, consistent with mechanical rather than biological transmission. The parameters obtained in this study were combined with data from studies of LSDV transmission and vector life history parameters to determine the basic reproduction number of LSDV in cattle mediated by each of the model species. This reproduction number was highest for Stomoxys calcitrans (19.1), followed by C. nubeculosus (7.1) and Ae. aegypti (2.4), indicating that these three species are potentially efficient transmitters of LSDV; this information can be used to inform LSD control programs.IMPORTANCE Lumpy skin disease virus (LSDV) causes a severe systemic disease characterized by cutaneous nodules in cattle. LSDV is a rapidly emerging pathogen, having spread since 2012 into Europe and Russia and across Asia. The vector-borne nature of LSDV transmission is believed to have promoted this rapid geographic spread of the virus; however, a lack of quantitative evidence about LSDV transmission has hampered effective control of the disease during the current epidemic. Our research shows subclinical cattle play little part in virus transmission relative to clinical cattle and reveals a low probability of virus acquisition by insects at the preclinical stage. We have also calculated the reproductive number of different insect species, therefore identifying efficient transmitters of LSDV. This information is of utmost importance, as it will help to define epidemiological control measures during LSDV epidemics and of particular consequence in resource-poor regions where LSD vaccination may be less than adequate.

Unpicking the Secrets of African Swine Fever Viral Replication Sites.

African swine fever virus (ASFV) is a highly contagious pathogen which causes a lethal haemorrhagic fever in domestic pigs and wild boar. The large, double-stranded DNA virus replicates in perinuclear cytoplasmic replication sites known as viral factories. These factories are complex, multi-dimensional structures. Here we investigated the protein and membrane compartments of the factory using super-resolution and electron tomography. Click IT chemistry in combination with stimulated emission depletion (STED) microscopy revealed a reticular network of newly synthesized viral proteins, including the structural proteins p54 and p34, previously seen as a pleomorphic ribbon by confocal microscopy. Electron microscopy and tomography confirmed that this network is an accumulation of membrane assembly intermediates which take several forms. At early time points in the factory formation, these intermediates present as small, individual membrane fragments which appear to grow and link together, in a continuous progression towards new, icosahedral virions. It remains unknown how these membranes form and how they traffic to the factory during virus morphogenesis.

Green synthesis as a simple and rapid route to protein modified magnetic nanoparticles for use in the development of a fluorometric molecularly imprinted polymer-based assay for detection of myoglobin.

We have developed a low-cost molecularly imprinted polymer (MIP)-based fluorometric assay to directly quantify myoglobin in a biological sample. The assay uses a previously unreported method for the development of microwave-assisted rapid synthesis of aldehyde functionalized magnetic nanoparticles, in just 20 min. The aldehyde functionalized nanoparticles have an average size of 7.5 nm ± 1.8 and saturation magnetizations of 31.8 emu g-1 with near-closed magnetization loops, confirming their superparamagnetic properties. We have subsequently shown that protein tethering was possible to the aldehyde particles, with 0.25 ± 0.013 mg of myoglobin adsorbed to 20 mg of the nanomaterial. Myoglobin-specific fluorescently tagged MIP (F-MIP) particles were synthesized and used within the assay to capture myoglobin from a test sample. Excess F-MIP was removed from the sample using protein functionalized magnetic nanoparticles (Mb-SPION), with the remaining sample analyzed using fluorescence spectroscopy. The obtained calibration plot of myoglobin showed a linear correlation ranging from 60 pg ml-1 to 6 mg ml-1 with the limit of detection of 60 pg ml-1. This method was successfully used to detect myoglobin in spiked fetal calf serum, with a recovery rate of more than 93%.

Preparation of Cultured Cells Using High-Pressure Freezing and Freeze Substitution for Subsequent 2D or 3D Visualization in the Transmission Electron Microscope.

Transmission electron microscopy (TEM) is an invaluable technique used for imaging the ultrastructure of samples, and it is particularly useful when determining virus-host interactions at a cellular level. The environment inside a TEM is not favorable for biological material (high vacuum and high energy electrons). Also biological samples have little or no intrinsic electron contrast and rarely do they naturally exist in very thin sheets, as is required for optimum resolution in the TEM. To prepare these samples for imaging in the TEM therefore requires extensive processing which can alter the ultrastructure of the material. Here we describe a method which aims to minimize preparation artifacts by freezing the samples at high pressure to instantaneously preserve ultrastructural detail, then rapidly substituting the ice with resin to provide a firm matrix which can be cut into thin sections for imaging. Thicker sections of this material can also be imaged and reconstructed into 3D volumes using electron tomography.

Lumpy Skin Disease Is Characterized by Severe Multifocal Dermatitis With Necrotizing Fibrinoid Vasculitis Following Experimental Infection.

Lumpy skin disease is a high-consequence disease in cattle caused by infection with the poxvirus lumpy skin disease virus (LSDV). The virus is endemic in most countries in Africa and an emerging threat to cattle populations in Europe and Asia. As LSDV spreads into new regions, it is important that signs of disease are recognized promptly by animal caregivers. This study describes the gross, microscopic, and ultrastructural changes that occur over time in cattle experimentally challenged with LSDV. Four calves were inoculated with wildtype LSDV and monitored for 19 to 21 days. At 7 days after inoculation, 2 of the 4 cattle developed multifocal cutaneous nodules characteristic of LSD. Some lesions displayed a targetoid appearance. Histologically, intercellular and intracellular edema was present in the epidermis of some nodules. Occasional intracytoplasmic inclusion bodies were identified in keratinocytes. More severe and consistent changes were present in the dermis, with marked histiocytic inflammation and necrotizing fibrinoid vasculitis of dermal vessels, particularly the deep dermal plexus. Chronic lesions consisted of full-thickness necrosis of the dermis and epidermis. Lesions in other body organs were not a major feature of LSD in this study, highlighting the strong cutaneous tropism of this virus. Immunohistochemistry and electron microscopy identified LSDV-infected histiocytes and fibroblasts in the skin nodules of affected cattle. This study highlights the noteworthy lesions of LSDV and how they develop over time.

Discrete Virus Factories Form in the Cytoplasm of Cells Coinfected with Two Replication-Competent Tagged Reporter Birnaviruses That Subsequently Coalesce over Time.

The Birnaviridae family, responsible for major economic losses to poultry and aquaculture, is composed of nonenveloped viruses with a segmented double-stranded RNA (dsRNA) genome that replicate in discrete cytoplasmic virus factories (VFs). Reassortment is common; however, the underlying mechanism remains unknown given that VFs may act as a barrier to genome mixing. In order to provide new information on VF trafficking during dsRNA virus coinfection, we rescued two recombinant infectious bursal disease viruses (IBDVs) of strain PBG98 containing either a split GFP11 or a tetracysteine (TC) tag fused to the VP1 polymerase (PBG98-VP1-GFP11 and PBG98-VP1-TC). DF-1 cells transfected with GFP1-10 prior to PBG98-VP1-GFP11 infection or stained with a biarsenical derivative of the red fluorophore resorufin (ReAsH) following PBG98-VP1-TC infection, had green or red foci in the cytoplasm, respectively, that colocalized with VP3 and dsRNA, consistent with VFs. The average number of VFs decreased from a mean of 60 to 5 per cell between 10 and 24 h postinfection (hpi) (P < 0.0001), while the average area increased from 1.24 to 45.01 µm2 (P < 0.0001), and live cell imaging revealed that the VFs were highly dynamic structures that coalesced in the cytoplasm. Small VFs moved faster than large (average 0.57 µm/s at 16 hpi compared to 0.22 µm/s at 22 hpi), and VF coalescence was dependent on an intact microtubule network and actin cytoskeleton. During coinfection with PBG98-VP1-GFP11 and PBG98-VP1-TC viruses, discrete VFs initially formed from each input virus that subsequently coalesced 10 to 16 hpi, and we speculate that Birnaviridae reassortment requires VF coalescence.IMPORTANCE Reassortment is common in viruses with segmented double-stranded RNA (dsRNA) genomes. However, these viruses typically replicate within discrete cytoplasmic virus factories (VFs) that may represent a barrier to genome mixing. We generated the first replication competent tagged reporter birnaviruses, infectious bursal disease viruses (IBDVs) containing a split GFP11 or tetracysteine (TC) tag and used the viruses to track the location and movement of IBDV VFs, in order to better understand the intracellular dynamics of VFs during a coinfection. Discrete VFs initially formed from each virus that subsequently coalesced from 10 h postinfection. We hypothesize that VF coalescence is required for the reassortment of the Birnaviridae This study provides new information that adds to our understanding of dsRNA virus VF trafficking.

The Porcine Deltacoronavirus Replication Organelle Comprises Double-Membrane Vesicles and Zippered Endoplasmic Reticulum with Double-Membrane Spherules.

Porcine deltacoronavirus (PDCoV) was first identified in Hong Kong in 2012 from samples taken from pigs in 2009. PDCoV was subsequently identified in the USA in 2014 in pigs with a history of severe diarrhea. The virus has now been detected in pigs in several countries around the world. Following the development of tissue culture adapted strains of PDCoV, it is now possible to address questions regarding virus-host cell interactions for this genera of coronavirus. Here, we presented a detailed study of PDCoV-induced replication organelles. All positive-strand RNA viruses induce the rearrangement of cellular membranes during virus replication to support viral RNA synthesis, forming the replication organelle. Replication organelles for the Alpha-, Beta-, and Gammacoronavirus genera have been characterized. All coronavirus genera induced the formation of double-membrane vesicles (DMVs). In addition, Alpha- and Betacoronaviruses induce the formation of convoluted membranes, while Gammacoronaviruses induce the formation of zippered endoplasmic reticulum (ER) with tethered double-membrane spherules. However, the structures induced by Deltacoronaviruses, particularly the presence of convoluted membranes or double-membrane spherules, are unknown. Initially, the dynamics of PDCoV strain OH-FD22 replication were assessed with the onset of viral RNA synthesis, protein synthesis, and progeny particle release determined. Subsequently, virus-induced membrane rearrangements were identified in infected cells by electron microscopy. As has been observed for all other coronaviruses studied to date, PDCoV replication was found to induce the formation of double-membrane vesicles. Significantly, however, PDCoV replication was also found to induce the formation of regions of zippered endoplasmic reticulum, small associated tethered vesicles, and double-membrane spherules. These structures strongly resemble the replication organelle induced by avian Gammacoronavirus infectious bronchitis virus.

Evaluation of Molecularly Imprinted Polymers as Synthetic Virus Neutralizing Antibody Mimics.

Rapid development of antibody-based therapeutics are crucial to the agenda of innovative manufacturing of macromolecular therapies to combat emergent diseases. Although highly specific, antibody therapies are costly to produce. Molecularly imprinted polymers (MIPs) constitute a rapidly-evolving class of antigen-recognition materials that act as synthetic antibodies. We report here on the virus neutralizing capacity of hydrogel-based MIPs. We produced MIPs using porcine reproductive and respiratory syndrome virus (PRRSV-1), as a model mammalian virus. Assays were performed to evaluate the specificity of virus neutralization, the effect of incubation time and MIP concentration. Polyacrylamide and N-hydroxymethylacrylamide based MIPs produced a highly significant reduction in infectious viral titer recovered after treatment, reducing it to the limit of detection of the assay. MIP specificity was tested by comparing their neutralizing effects on PRRSV-1 to the effects on the unrelated bovine viral diarrhea virus-1; no significant cross-reactivity was observed. The MIPs demonstrated effective virus neutralization in just 2.5 min and their effect was concentration dependent. These data support the further evaluation of MIPs as synthetic antibodies as a novel approach to the treatment of viral infection.

Carbenoxolone-mediated cytotoxicity inhibits Vaccinia virus replication in a human keratinocyte cell line.

The re-emergence of poxviral zoonotic infections and the threat of bioterrorism emphasise the demand for effective antipoxvirus therapies. Here, we show that carbenoxolone, a pharmacological inhibitor of gap junction function and a compound widely used in cell culture, is capable of hindering the replication of Vaccinia virus, the prototypical poxvirus, in a gap junction-independent manner in a human keratinocyte cell line. Viral protein synthesis occurs in the presence of carbenoxolone but infectious virion formation is minimal, indicating that carbenoxolone blocks viral morphogenesis. Initial viability tests suggested that carbenoxolone was not toxic to cells. However, electron microscopic analysis of carbenoxolone treated cells revealed that it alters the cellular endomembrane system. This widespread ultrastructural damage prevents Vaccinia virus virion assembly. These results strengthen the need for thorough characterisation of the effects of antiviral compounds on the cellular ultrastructure.

Infectious Bronchitis Virus Nonstructural Protein 4 Alone Induces Membrane Pairing.

Positive-strand RNA viruses, such as coronaviruses, induce cellular membrane rearrangements during replication to form replication organelles allowing for efficient viral RNA synthesis. Infectious bronchitis virus (IBV), a pathogenic avian Gammacoronavirus of significant importance to the global poultry industry, has been shown to induce the formation of double membrane vesicles (DMVs), zippered endoplasmic reticulum (zER) and tethered vesicles, known as spherules. These membrane rearrangements are virally induced; however, it remains unclear which viral proteins are responsible. In this study, membrane rearrangements induced when expressing viral non-structural proteins (nsps) from two different strains of IBV were compared. Three non-structural transmembrane proteins, nsp3, nsp4, and nsp6, were expressed in cells singularly or in combination and the effects on cellular membranes investigated using electron microscopy and electron tomography. In contrast to previously studied coronaviruses, IBV nsp4 alone is necessary and sufficient to induce membrane pairing; however, expression of the transmembrane proteins together was not sufficient to fully recapitulate DMVs. This indicates that although nsp4 is able to singularly induce membrane pairing, further viral or host factors are required in order to fully assemble IBV replicative structures. This study highlights further differences in the mechanism of membrane rearrangements between members of the coronavirus family.

Preparation of cultured cells using high-pressure freezing and freeze substitution for subsequent 2D or 3D visualization in the transmission electron microscope.

Transmission electron microscopy (TEM) is an invaluable technique used for imaging the ultrastructure of samples and it is particularly useful when determining virus-host interactions at a cellular level. The environment inside a TEM is not favorable for biological material (high vacuum and high energy electrons). Also biological samples have little or no intrinsic electron contrast, and rarely do they naturally exist in very thin sheets, as is required for optimum resolution in the TEM. To prepare these samples for imaging in the TEM therefore requires extensive processing which can alter the ultrastructure of the material. Here we describe a method which aims to minimize preparation artifacts by freezing the samples at high pressure to instantaneously preserve ultrastructural detail, then rapidly substituting the ice and infiltrating with resin to provide a firm matrix which can be cut into thin sections for imaging. Thicker sections of this material can also be imaged and reconstructed into 3D volumes using electron tomography.

Spherules and IBV.

Infectious bronchitis virus (IBV) is an economically important virus infecting chickens, causing large losses to the poultry industry globally. While vaccines are available, there is a requirement for novel vaccine strategies due to high strain variation and poor cross-protection. This requires a more detailed understanding of virus-host cell interactions to identify candidates for targeted virus attenuation. One key area of research in the positive sense RNA virus field, due to its central role in virus replication, is the induction of cellular membrane rearrangements by this class of viruses for the assembly of virus replication complexes. In our recent work, we identified the structures induced by IBV during infection of cultured cells, as well as primary cells and ex vivo organ culture. We identified structures novel to the coronavirus family, which strongly resemble replication sites of other positive sense RNA viruses. We have begun to extend this work using recombinant IBVs, which are chimera of different virus strains to study the role of viral proteins in the induction of membrane rearrangements.

Infectious bronchitis virus generates spherules from zippered endoplasmic reticulum membranes.

UNLABELLED: Replication of positive-sense RNA viruses is associated with the rearrangement of cellular membranes. Previous work on the infection of tissue culture cell lines with the betacoronaviruses mouse hepatitis virus and severe acute respiratory syndrome coronavirus (SARS-CoV) showed that they generate double-membrane vesicles (DMVs) and convoluted membranes as part of a reticular membrane network. Here we describe a detailed study of the membrane rearrangements induced by the avian gammacoronavirus infectious bronchitis virus (IBV) in a mammalian cell line but also in primary avian cells and in epithelial cells of ex vivo tracheal organ cultures. In all cell types, structures novel to IBV infection were identified that we have termed zippered endoplasmic reticulum (ER) and spherules. Zippered ER lacked luminal space, suggesting zippering of ER cisternae, while spherules appeared as uniform invaginations of zippered ER. Electron tomography showed that IBV-induced spherules are tethered to the zippered ER and that there is a channel connecting the interior of the spherule with the cytoplasm, a feature thought to be necessary for sites of RNA synthesis but not seen previously for membrane rearrangements induced by coronaviruses. We also identified DMVs in IBV-infected cells that were observed as single individual DMVs or were connected to the ER via their outer membrane but not to the zippered ER. Interestingly, IBV-induced spherules strongly resemble confirmed sites of RNA synthesis for alphaviruses, nodaviruses, and bromoviruses, which may indicate similar strategies of IBV and these diverse viruses for the assembly of RNA replication complexes. IMPORTANCE: All positive-sense single-stranded RNA viruses induce rearranged cellular membranes, providing a platform for viral replication complex assembly and protecting viral RNA from cellular defenses. We have studied the membrane rearrangements induced by an important poultry pathogen, the gammacoronavirus infectious bronchitis virus (IBV). Previous work studying closely related betacoronaviruses identified double-membrane vesicles (DMVs) and convoluted membranes (CMs) derived from the endoplasmic reticulum (ER) in infected cells. However, the role of DMVs and CMs in viral RNA synthesis remains unclear because these sealed vesicles lack a means of delivering viral RNA to the cytoplasm. Here, we characterized structures novel to IBV infection: zippered ER and small vesicles tethered to the zippered ER termed spherules. Significantly, spherules contain a channel connecting their interior to the cytoplasm and strongly resemble confirmed sites of RNA synthesis for other positive-sense RNA viruses, making them ideal candidates for the site of IBV RNA synthesis.

Foot-and-mouth disease virus replicates only transiently in well-differentiated porcine nasal epithelial cells.

Three-dimensional (3D) porcine nasal mucosal and tracheal mucosal epithelial cell cultures were developed to analyze foot-and-mouth disease virus (FMDV) interactions with mucosal epithelial cells. The cells in these cultures differentiated and polarized until they closely resemble the epithelial layers seen in vivo. FMDV infected these cultures predominantly from the apical side, primarily by binding to integrin alphav beta6, in an Arg-Gly-Asp (RGD)-dependent manner. However, FMDV replicated only transiently without any visible cytopathic effect (CPE), and infectious progeny virus could be recovered only from the apical side. The infection induced the production of beta interferon (IFN-beta) and the IFN-inducible gene Mx1 mRNA, which coincided with the disappearance of viral RNA and progeny virus. The induction of IFN-beta mRNA correlated with the antiviral activity of the supernatants from both the apical and basolateral compartments. IFN-alpha mRNA was constitutively expressed in nasal mucosal epithelial cells in vitro and in vivo. In addition, FMDV infection induced interleukin 8 (IL-8) protein, granulocyte-macrophage colony-stimulating factor (GM-CSF), and RANTES mRNA in the infected epithelial cells, suggesting that it plays an important role in modulating the immune response.

The envelope of intracellular African swine fever virus is composed of a single lipid bilayer.

African swine fever virus (ASFV) is a member of a family of large nucleocytoplasmic DNA viruses that include poxviruses, iridoviruses, and phycodnaviruses. Previous ultrastructural studies of ASFV using chemical fixation and cryosectioning for electron microscopy (EM) have produced uncertainty over whether the inner viral envelope is composed of a single or double lipid bilayer. In this study we prepared ASFV-infected cells for EM using chemical fixation, cryosectioning, and high-pressure freezing. The appearance of the intracellular viral envelope was determined and compared to that of mitochondrial membranes in each sample. The best resolution of membrane structure was obtained with samples prepared by high-pressure freezing, and images suggested that the envelope of ASFV consisted of a single lipid membrane. It was less easy to interpret virus structure in chemically fixed or cryosectioned material, and in the latter case the virus envelope could be interpreted as having two membranes. Comparison of membrane widths in all three preparations indicated that the intracellular viral envelope of ASFV was not significantly different from the outer mitochondrial membrane (P < 0.05). The results support the hypothesis that the intracellular ASFV viral envelope is composed of a single lipid bilayer.