Spatial multiomic profiling reveals the novel polarization of foamy macrophages within necrotic granulomatous lesions developed in lungs of C3HeB/FeJ mice infected with Mycobacterium tuberculosis.

Infection with Mycobacterium tuberculosis leads to the development of tuberculosis (TB) with the formation of granulomatous lesions. Foamy macrophages (FM) are a hallmark of TB granulomas, because they provide the primary platform of M. tuberculosis proliferation and the main source of caseous necrosis. In this study, we applied spatial multiomic profiling to identify the signatures of FM within the necrotic granulomas developed in a mouse model resembling human TB histopathology. C3HeB/FeJ mice were infected with M. tuberculosis to induce the formation of necrotic granulomas in the lungs. Using laser microdissection, necrotic granulomas were fractionated into three distinct regions, including the central caseous necrosis, the rim containing FM, and the peripheral layer of macrophages and lymphocytes, and subjected to proteomic and transcriptomic analyses. Comparison of proteomic and transcriptomic analyses of three distinct granulomatous regions revealed that four proteins/genes are commonly enriched in the rim region. Immunohistochemistry confirmed the localization of identified signatures to the rim of necrotic granulomas. We also investigated the localization of the representative markers for M1 macrophages in granulomas because the signatures of the rim included M2 macrophage markers. The localization of both macrophage markers suggests that FM in necrotic granulomas possessed the features of M1 or M2 macrophages. Gene set enrichment analysis of transcriptomic profiling revealed the upregulation of genes related to M2 macrophage activation and mTORC1 signaling in the rim. These results will provide new insights into the process of FM biogenesis, leading to further understanding of the pathophysiology of TB granulomas.

Recurring large deletion in DRC1 (CCDC164) identified as causing primary ciliary dyskinesia in two Asian patients.

BACKGROUND: Primary ciliary dyskinesia (PCD) is a relatively rare autosomal recessive or X-linked disorder affecting ciliary function. In the set of causative genes, however, predominant pathogenic variants remain unknown in Asia. METHOD: A diagnosis of PCD was made following a modern comprehensive testing including genetic analysis; targeted resequencing for screening variants, and Sanger sequencing for determination of the breakpoints, with an additional review of databases to calculate the deletion frequency. A multiplexed PCR-based detection method has also been developed. RESULTS: We ascertained a 50-year-old Japanese male who had been diagnosed with diffuse panbronchiolitis (DPB), but refractory to macrolide therapy. We reevaluated the case and identified a large homozygous deletion spanning exons 1 to 4 of the DRC1 and determined the breakpoints (NM_145038.4: c.1-3952_540 + 1331del27748-bp). In the PCD cohort at the University of North Carolina, we found a female PCD patient of Korean descent harboring the same homozygous deletion. From the Invitae testing cohort, we extracted four carriers of the same deletion among 965 Asian individuals, whereas no deletion was found in the 23,951 non-Asians. CONCLUSION: We speculate that the DRC1 deletion is a recurrent or perhaps founder mutation in Asians. The simple PCR method could be a useful screening tool.

Experimental piscine alphavirus RNA recombination in vivo yields both viable virus and defective viral RNA.

RNA recombination in non-segmented RNA viruses is important for viral evolution and documented for several virus species through in vitro studies. Here we confirm viral RNA recombination in vivo using an alphavirus, the SAV3 subtype of Salmon pancreas disease virus. The virus causes pancreas disease in Atlantic salmon and heavy losses in European salmonid aquaculture. Atlantic salmon were injected with a SAV3 6K-gene deleted cDNA plasmid, encoding a non-viable variant of SAV3, together with a helper cDNA plasmid encoding structural proteins and 6K only. Later, SAV3-specific RNA was detected and recombination of viral RNA was confirmed. Virus was grown from plasmid-injected fish and shown to infect and cause pathology in salmon. Subsequent cloning of PCR products confirming recombination, documented imprecise homologous recombination creating RNA deletion variants in fish injected with cDNA plasmid, corresponding with deletion variants previously found in SAV3 from the field. This is the first experimental documentation of alphavirus RNA recombination in an animal model and provides new insight into the production of defective virus RNA.

In vitro adaptation of SAV3 in cell culture correlates with reduced in vivo replication capacity and virulence to Atlantic salmon (Salmo salar L.) parr.

Salmonid alphavirus (SAV) is the causative agent of pancreas disease affecting Atlantic salmon and rainbow trout and causes a major burden to the aquaculture industry. This study describes a Norwegian subtype SAV3 virus isolate (SAV3-H10) subjected to serial passages in Chinook salmon embryo cells (CHSE-214) followed by Asian Grouper skin cells (AGK). Two passages from CHSE and one after transfer to AGK cells were chosen for further investigation, based on variation in degree and development of cytopathic effect (CPE). After plaque purification, several in vitro studies were performed. Cell viability after infection, viral replication and ability to cause morphological changes in CHSE and AGK cells was studied for the three isolates. The AGK-transferred isolate was identified with the strongest abilities to reduce cell viability, replicate more and cause more CPE in cell culture when compared with the early and late CHSE-grown isolates. Subsequently, the isolates were tested in an experimental fish challenge, showing higher viral load and higher pathological score for the least cell-cultured isolate. Full-length sequencing of the viral genome of the three isolates revealed divergence in four amino acid positions and the AGK-grown isolate also had a 3 nt deletion in the 3'UTR. In conclusion, we show that cell culture of SAV3-H10 selects for strains inducing earlier CPE in vitro with increased viral replication. In vivo, the effect is reversed, with lower replication levels and lower pathology scores in target organs. This study outlines a path to identify potential virulence motifs of SAV3.

Sequence analysis and identification of new isoform of EP4 receptors in different atlantic salmon tissues (Salmo salar L.) and its role in PGE2 induced immunomodulation in vitro.

PGE2 plays an important role in a broad spectrum of physiological and pathological processes mediated through a membrane-bound G protein-coupled receptor (GPCR) called EP receptor. In mammals, four subtypes of EP receptor (EP 1-4) are identified and each of them functions through different signal transduction pathways. Orthologous EP receptors have also been identified in other non-mammalian species, such as chicken and zebrafish. EP4 is the only identified PGE2 receptor to date in Atlantic salmon but its tissue distribution and function have not been studied in any detail. In this study, we first sequenced EP4 receptor in different tissues and found that the presence of the 3nt deletion in the 5' untranslated region was accompanied by silent mutation at nt 668. While attempting to amplify the same sequence in TO cells (an Atlantic salmon macrophage-like cell line), we failed to obtain the full-length product. Further investigation revealed different isoform of EP4 receptor in TO cells and we subsequently documented its presence in different Atlantic salmon tissues. These two isoforms of EP4 receptor share high homology in their first half of sequence but differ in the second half part with several deletion segments though the final length of coding sequence is the same for two isoforms. We further studied the immunomodulation effect of PGE2 in TO cells and found that PGE2 inhibited the induction of CXCL-10, CCL-4, IL-8 and IL-1ß genes expression in a time dependent manner and without cAMP upregulation.

Specific nucleotides at the 3'-terminal promoter of viral hemorrhagic septicemia virus are important for virulence in vitro and in vivo.

Viral hemorrhagic septicemia virus (VHSV), a member of the Novirhabdovirus genus, contains an 11-nucleotide conserved sequence at the terminal 3'- and 5'-untranslated regions (UTRs) that are complementary. To study the importance of nucleotides in the 3'-UTR of VHSV for replication of novirhabdoviruses, we performed site-directed mutagenesis of selected residues at the 3'-terminus and generated mutant viruses using a reverse genetics approach. Assessment of growth kinetics and in vitro real-time cytopathogenicity studies showed that the order of two nucleotides (A4G5) of the 3'-terminus of VHSV directly affects growth kinetics in vitro. The mutant A4G-G5A virus has reduced total positive-strand RNA synthesis efficiency (51% of wild-type) at 48h post-transfection and 70h delay in causing complete cytopathic effect in susceptible fish cells, as compared to the WT-VHSV. Furthermore, when the A4G-G5A virus was used to challenge zebrafish, it exhibited reduced pathogenicity (54% lower end-point mortality) compared to the WT-VHSV. From these studies, we infer that specific residues in the 3'-UTR of VHSV have a promoter function and are essential to modulate the virulence in cells and pathogenicity in fish.

Modification of a salmonid alphavirus replicon vector for enhanced expression of heterologous antigens.

A salmonid alphavirus (SAV) replicon has been developed to express heterologous antigens but protein production was low to modest compared with terrestrial alphavirus replicons. In this study, we have compared several modifications to a SAV replicon construct and analysed their influence on foreign gene expression. We found that an insertion of a translational enhancer consisting of the N-terminal 102 nt of the capsid gene, together with a nucleotide sequence encoding the foot-and-mouth disease virus (FMDV) 2A peptide, caused a significant increase in EGFP reporter gene expression. The importance of fusing a hammerhead (HH) ribozyme sequence at the 5' end of the viral genome was also demonstrated. In contrast, a hepatitis D virus ribozyme (HDV-RZ) sequence placed at the 3' end did not augment expression of inserted genes. Taken together, we have developed a platform for optimized antigen production, which can be applied for immunization of salmonid fish in the future.

A 6K-deletion variant of salmonid alphavirus is non-viable but can be rescued through RNA recombination.

Pancreas disease (PD) of Atlantic salmon is an emerging disease caused by Salmonid alphavirus (SAV) which mainly affects salmonid aquaculture in Western Europe. Although genome structure of SAV has been characterized and each individual viral protein has been identified, the role of 6K protein in viral replication and infectivity remains undefined. The 6K protein of alphaviruses is a small and hydrophobic protein which is involved in membrane permeabilization, protein processing and virus budding. Because these common features are shared across many viral species, they have been named viroporins. In the present study, we applied reverse genetics to generate SAV3 6K-deleted (Δ6K) variant and investigate the role of 6K protein. Our findings show that the 6K-deletion variant of salmonid alphavirus is non-viable. Despite viral proteins of Δ6K variant are detected in the cytoplasm by immunostaining, they are not found on the cell surface. Further, analysis of viral proteins produced in Δ6K cDNA clone transfected cells using radioimmunoprecipitation (RIPA) and western blot showed a protein band of larger size than E2 of wild-type SAV3. When Δ6K cDNA was co-transfected with SAV3 helper cDNA encoding the whole structural genes including 6K, the infectivity was rescued. The development of CPE after co-transfection and resolved genome sequence of rescued virus confirmed full-length viral genome being generated through RNA recombination. The discovery of the important role of the 6K protein in virus production provides a new possibility for the development of antiviral intervention which is highly needed to control SAV infection in salmonids.

Gene expression studies of host response to Salmonid alphavirus subtype 3 experimental infections in Atlantic salmon.

Salmonid alphavirus subtype-3 (SAV-3) infection in Atlantic salmon is exclusively found in Norway. The salmonid alphaviruses have been well characterized at the genome level but there is limited information about the host-pathogen interaction phenomena. This study was undertaken to characterize the replication and spread of SAV-3 in internal organs of experimentally infected Atlantic salmon and the subsequent innate and adaptive immune responses. In addition, suitability of a cohabitation challenge model for this virus was also examined. Groups of fish were infected by intramuscular injection (IM), cohabited (CO) or kept uninfected in a separate tank. Samples of pancreas, kidney, spleen, heart and skeletal muscles were collected at 2, 4 and 8 weeks post infection (wpi). Pathological changes were assessed by histology concurrently with viral loads and mRNA expression of immune genes by real time RT-PCR. Pathological changes were only observed in the pancreas and heart (target organs) of both IM and CO groups, with changes appearing first in the pancreas (2 wpi) in the former. Lesions with increasing severity over time coincided with high viral loads despite significant induction of IFN-α, Mx and ISG15. IFN-γ and MHC-I were expressed in all tissues examined and their induction appeared in parallel with that of IL-10. Inflammatory genes TNF-α, IL-12 and IL-8 were only induced in the heart during pathology while T cell-related genes CD3ε, CD4, CD8, TCR-α and MHC-II were expressed in target organs at 8 wpi. These findings suggest that the onset of innate responses came too late to limit virus replication. Furthermore, SAV-3 infections in Atlantic salmon induce Th1/cytotoxic responses in common with other alphaviruses infecting higher vertebrates. Our findings demonstrate that SAV-3 can be transmitted via the water making it suitable for a cohabitation challenge model.

Alpha interferon and not gamma interferon inhibits salmonid alphavirus subtype 3 replication in vitro.

Salmonid alphavirus (SAV) is an emerging virus in salmonid aquaculture, with SAV-3 being the only subtype found in Norway. Until now, there has been little focus on the alpha interferon (IFN-alpha)-induced antiviral responses during virus infection in vivo or in vitro in fish. The possible involvement of IFN-gamma in the response to SAV-3 is also not known. In this study, the two IFNs were cloned and expressed as recombinant proteins (recombinant IFN-alpha [rIFN-alpha] and rIFN-gamma) and used for in vitro studies. SAV-3 infection in a permissive salmon cell line (TO cells) results in IFN-alpha and IFN-stimulated gene (ISG) mRNA upregulation. Preinfection treatment (4 to 24 h prior to infection) with salmon rIFN-alpha induces an antiviral state that inhibits the replication of SAV-3 and protects the cells against virus-induced cytopathic effects (CPE). The antiviral state coincides with a strong expression of Mx and ISG15 mRNA and Mx protein expression. When rIFN-alpha is administered at the time of infection and up to 24 h postinfection, virus replication is not inhibited, and cells are not protected against virus-induced CPE. By 40 h postinfection, the alpha subunit of eukaryotic initiation factor 2 (eIF2alpha) is phosphorylated concomitant with the expression of the E2 protein as assessed by Western blotting. Postinfection treatment with rIFN-alpha results in a moderate reduction in E2 expression levels in accordance with a moderate downregulation of cellular protein synthesis, an approximately 65% reduction by 60 h postinfection. rIFN-gamma has only a minor inhibitory effect on SAV-3 replication in vitro. SAV-3 is sensitive to the preinfection antiviral state induced by rIFN-alpha, while postinfection antiviral responses or postinfection treatment with rIFN-alpha is not able to limit viral replication.

The interferon response is involved in nervous necrosis virus acute and persistent infection in zebrafish infection model.

Betanodavirus, a small positive-sense bipartite RNA virus notoriously affecting marine aquaculture worldwide has been extensively studied in vitro. However, impending studies in elucidating virus-host interactions have been limiting due to the lack of appropriate animal disease models. Therefore, in this study, we have attempted to successfully establish NNV infection in zebrafish (Danio rerio) showing typical NNV symptoms and which could potentially serve as an in vivo model for studying virus pathogenesis. Zebrafish being already a powerful research tool in developmental biology and having its genome completely sequenced by the end of 2007 would expedite NNV research. We have observed viral titers peaked at 3 days post-infection and histological study showing lesions in brain tissues similar to natural host infection. Further, we used this infection model to study the acute and persistence infection during NNV infection. Interestingly, RT-PCR and immunoblotting assays revealed that the acute infection in larvae and juveniles is largely due to inactive interferon response as opposed to activated innate immune response during persistent infection in adult stage. This study is the first to demonstrate NNV infection of zebrafish, which could serve as a potential animal model to study virus pathogenesis and neuron degeneration research.