Application of CRISPR/Cas9 for Rapid Genome Editing of Pseudorabies Virus and Bovine Herpesvirus-1.

The CRISPR/Cas9 system is widely used to manipulate viral genomes. Although Alphaherpesvirinae genomes are large and complicated to edit, in recent years several Pseudorabies virus (PRV) mutants have been successfully generated using the CRISPR/Cas9 system. However, the application of CRISPR/Cas9 editing on another member of alpha herpesviruses, bovine herpesvirus-1 (BHV-1), is rarely reported. This paper reports a rapid and straightforward approach to manipulating herpesviruses genome using CRISPR/Cas9. The recombinant plasmids contained the left and right arm of the thymidine kinase (TK) gene of PRV or of the glycoprotein I (gI) and glycoprotein E (gE) of BHV-1. Upon the cleavage of the TK or gIgE gene by Cas9 protein, this was replaced by the enhanced green fluorescence protein (eGFP) by homologous recombination. With this approach, we generated recombinant TK-/eGFP+ PRV and gIgE-/eGFP+ BHV-1 mutants and then proceeded to characterize their biological activities in vitro and in vivo. In conclusion, we showed that alpha herpesvirus, including PRV and BHV-1, can be rapidly edited using the CRISPR/Cas9 approach paving the way to the development of animal herpesvirus vaccines.

Immunoadjuvant efficacy of CpG plasmids for H9N2 avian influenza inactivated vaccine in chickens with maternal antibodies.

Maternal-derived antibodies (MDAs) are one of reasons why vaccination with the H9N2 inactivated whole virus (IWV) vaccine failed in poultry. Unmethylated CpG motif-containing oligodeoxynucleotides (CpG ODN) shows great potential to overcome MDAs interference in mammals, but whether it has similar characteristics in poultry is still unknown. In the present study, different classes and various copies of CpG ODN motifs were cloned into two different plasmids (pCDNA3.1 or T vector). Immunomodulatory activities and immunoadjuvant efficacy of these CpG ODN plasmids were tested in vitro and in vivo in the presence of passively transferred antibodies (PTAs) that were used to mimic MDAs. Results showed that the T vector enriched with 30 copies of CpG-A ODN and 20 copies of CpG-B ODN (T-CpG-AB) significantly up-regulated mRNA expression of chicken-interferon-α (ch-IFN-α), chicken-interferon-ß (ch-IFN-ß) and chicken-interleukin-12 protein 40 (ch-IL-12p40). When administered as adjuvant of the H9N2 IWV vaccine, the minimal dose of T-CpG-AB plasmid was 30 µg per one-day-old chicken, which could induce strong humoral immune responses in the presence of PTAs. Furthermore, T-CpG-AB plasmid-based vaccine triggered both strong humoral immune responses and cytokines expression in the presence of PTAs in chickens. Overall, our findings suggest that T-CpG-AB plasmid can be an excellent adjuvant candidate for the H9N2 IWV vaccine to overcome MDAs interference in chickens.

Efficacy of a recombinant turkey herpesvirus (H9) vaccine against H9N2 avian influenza virus in chickens with maternal-derived antibodies.

Although vaccines have been widely used for many years, they have failed to control H9N2 avian influenza virus (AIV) in the field in China. The high level of maternal-derived antibodies (MDAs) against H9N2 virus contributes to the H9N2 influenza vaccine failure in poultry. The study aimed to generate a new vaccine to overcome MDAs interference in H9N2 vaccination in chickens. We used turkey herpesvirus (HVT) as a vaccine vector to express H9 hemagglutinin (HA) proteins. The recombinant HVT expressing H9 HA proteins (rHVT-H9) was successfully generated and characterized in primary chicken embryonic fibroblasts (CEFs). Western blot and indirect immunofluorescence assay (IFA) showed that the rHVT-H9 consistently expressed HA proteins. In addition, the rHVT-H9 had similar growth kinetics to the parent HVT. Preliminary animal experiments showed that compared to the conventional inactivated whole virus (IWV) vaccine, the rHVT-H9 stimulated robust humoral immunity in chickens with passively transferred antibodies (PTAs) that were used to mimic MDAs. Transmission experiments showed that the rHVT-H9 induced both humoral and cellular immunity in chickens with PTAs. Furthermore, we used mathematical models to quantify the vaccine's efficacy in preventing the transmission of H9N2 AIV. The results showed that the rHVT-H9 reduced the virus shedding period and decreased the reproduction ratio (R) value in chickens with PTAs after homologous challenge. However, the vaccination in this trial did not yet bring R < 1. In summary, we generated a new rHVT-H9 vaccine, which stimulated strong humoral and cellular immunity, reducing virus shedding and transmission of H9N2 AIV even in the presence of PTAs in chickens.

Occurrence of foamy macrophages during the innate response of zebrafish to trypanosome infections.

A tightly regulated innate immune response to trypanosome infections is critical to strike a balance between parasite control and inflammation-associated pathology. In this study, we make use of the recently established Trypanosoma carassii infection model in larval zebrafish to study the early response of macrophages and neutrophils to trypanosome infections in vivo. We consistently identified high- and low-infected individuals and were able to simultaneously characterise their differential innate response. Not only did macrophage and neutrophil number and distribution differ between the two groups, but also macrophage morphology and activation state. Exclusive to high-infected zebrafish, was the occurrence of foamy macrophages characterised by a strong pro-inflammatory profile and potentially associated with an exacerbated immune response as well as susceptibility to the infection. To our knowledge, this is the first report of the occurrence of foamy macrophages during an extracellular trypanosome infection.

Differences in growth of Trypanoplasma borreli in carp serum is dependent on transferrin genotype.

Kinetoplastid parasites require transferrin (Tf), being the main source of iron, for growth and multiplication. This group of parasites developed a unique receptor-mediated system for acquiring host Tf which bears no structural homology with the host transferrin receptor. Trypanoplasma borreli, a blood parasite of common carp, probably uses a similar mechanism to sequester iron from host transferrin. In this study, we demonstrate a critical role of Tf for parasite growth. For in vitro studies we isolated and purified Tf from carp homozygous for the D or G allele of Tf. We obtained Tf-depleted serum using specific antibodies to carp Tf and studied gene expression in vivo during T. borreli infection with Real Time-quantitative PCR. We demonstrate that T. borreli cannot survive in medium supplemented with Tf-depleted serum while reconstitution with Tf restores normal growth. The critical role of Tf for parasite survival was shown in incomplete medium (medium without serum): addition of purified Tf significantly increased parasite survival. We also demonstrate that Tf polymorphism has a significant impact on T. borreli multiplication. Cultured parasites die more quickly in an environment containing D-typed Tf, as compared to medium with G-typed Tf. Gene expression during T. borreli infection in carp did not show an acute phase response. We could, however, observe an increased transcription of Tf in the head kidney, which may be associated with an immunological function of the Tf protein.

Inhibition of anti-viral stress granule formation by coronavirus endoribonuclease nsp15 ensures efficient virus replication.

Cytoplasmic stress granules (SGs) are generally triggered by stress-induced translation arrest for storing mRNAs. Recently, it has been shown that SGs exert anti-viral functions due to their involvement in protein synthesis shut off and recruitment of innate immune signaling intermediates. The largest RNA viruses, coronaviruses, impose great threat to public safety and animal health; however, the significance of SGs in coronavirus infection is largely unknown. Infectious Bronchitis Virus (IBV) is the first identified coronavirus in 1930s and has been prevalent in poultry farm for many years. In this study, we provided evidence that IBV overcomes the host antiviral response by inhibiting SGs formation via the virus-encoded endoribonuclease nsp15. By immunofluorescence analysis, we observed that IBV infection not only did not trigger SGs formation in approximately 80% of the infected cells, but also impaired the formation of SGs triggered by heat shock, sodium arsenite, or NaCl stimuli. We further demonstrated that the intrinsic endoribonuclease activity of nsp15 was responsible for the interference of SGs formation. In fact, nsp15-defective recombinant IBV (rIBV-nsp15-H238A) greatly induced the formation of SGs, along with accumulation of dsRNA and activation of PKR, whereas wild type IBV failed to do so. Consequently, infection with rIBV-nsp15-H238A strongly triggered transcription of IFN-ß which in turn greatly affected rIBV-nsp15-H238A replication. Further analysis showed that SGs function as an antiviral hub, as demonstrated by the attenuated IRF3-IFN response and increased production of IBV in SG-defective cells. Additional evidence includes the aggregation of pattern recognition receptors (PRRs) and signaling intermediates to the IBV-induced SGs. Collectively, our data demonstrate that the endoribonuclease nsp15 of IBV interferes with the formation of antiviral hub SGs by regulating the accumulation of viral dsRNA and by antagonizing the activation of PKR, eventually ensuring productive virus replication. We further demonstrated that nsp15s from PEDV, TGEV, SARS-CoV, and SARS-CoV-2 harbor the conserved function to interfere with the formation of chemically-induced SGs. Thus, we speculate that coronaviruses employ similar nsp15-mediated mechanisms to antagonize the host anti-viral SGs formation to ensure efficient virus replication.

ß-Glucan-Induced Immuno-Modulation: A Role for the Intestinal Microbiota and Short-Chain Fatty Acids in Common Carp.

Dietary supplementation of fish with ß-glucans has been commonly associated with immunomodulation and generally accepted as beneficial for fish health. However, to date the exact mechanisms of immunomodulation by ß-glucan supplementation in fish have remained elusive. In mammals, a clear relation between high-fibre diets, such as those including ß-glucans, and diet-induced immunomodulation via intestinal microbiota and associated metabolites has been observed. In this study, first we describe by 16S rRNA sequencing the active naive microbiota of common carp intestine. Based on the abundance of the genus Bacteroides, well known for their capacity to degrade and ferment carbohydrates, we hypothesize that common carp intestinal microbiota could ferment dietary ß-glucans. Indeed, two different ß-glucan preparations (curdlan and MacroGard®) were both fermented in vitro, albeit with distinct fermentation dynamics and distinct production of short-chain fatty acids (SCFA). Second, we describe the potential immunomodulatory effects of the three dominant SCFAs (acetate, butyrate, and propionate) on head kidney leukocytes, showing effects on both nitric oxide production and expression of several cytokines (il-1b, il-6, tnfα, and il-10) in vitro. Interestingly, we also observed a regulation of expression of several gpr40L genes, which were recently described as putative SCFA receptors. Third, we describe how a single in vivo oral gavage of carp with MacroGard® modulated simultaneously, the expression of several pro-inflammatory genes (il-1b, il-6, tnfα), type I IFN-associated genes (tlr3.1, mx3), and three specific gpr40L genes. The in vivo observations provide indirect support to our in vitro data and the possible role of SCFAs in ß-glucan-induced immunomodulation. We discuss how ß-glucan-induced immunomodulatory effects can be explained, at least in part, by fermentation of MacroGard® by specific bacteria, part of the naive microbiota of common carp intestine, and how a subsequent production of SFCAs could possibly explain immunomodulation by ß-glucan via SCFA receptors present on leukocytes.

Development of a Reverse Genetic System to Generate Recombinant Chimeric Tacaribe Virus that Expresses Junín Virus Glycoproteins.

Mammarenaviruses are enveloped and segmented negative-stranded RNA viruses that comprise several pathogenic members associated with severe human hemorrhagic fevers. Tacaribe virus (TCRV) is the prototype for the New World group of mammarenaviruses and is not only naturally attenuated but also phylogenetically and antigenically related to all South American pathogenic mammarenaviruses, particularly the Junín virus (JUNV), which is the etiological agent of Argentinian hemorrhagic fever (AHF). Moreover, since TCRV protects guinea pigs and non-human primates from lethal challenges with pathogenic strains of JUNV, it has already been considered as a potential live-attenuated virus vaccine candidate against AHF. Here, we report the development of a reverse genetic system that relies on T7 polymerase-driven intracellular expression of the complementary copy (antigenome) of both viral S and L RNA segments. Using this approach, we successfully recovered recombinant TCRV (rTCRV) that displayed growth properties resembling those of authentic TCRV. We also generated a chimeric recombinant TCRV expressing the JUNV glycoproteins, which propagated similarly to wild-type rTCRV. Moreover, a controlled modification within the S RNA 5' non-coding terminal sequence diminished rTCRV propagation in a cell-type dependent manner, giving rise to new perspectives where the incorporation of additional attenuation markers could contribute to develop safe rTCRV-based vaccines against pathogenic mammarenaviruses.

Transcriptome sequencing supports a conservation of macrophage polarization in fish.

Mammalian macrophages can adopt polarization states that, depending on the exact stimuli present in their extracellular environment, can lead to very different functions. Although these different polarization states have been shown primarily for macrophages of humans and mice, it is likely that polarized macrophages with corresponding phenotypes exist across mammals. Evidence of functional conservation in macrophages from teleost fish suggests that the same, or at least comparable polarization states should also be present in teleosts. However, corresponding transcriptional profiles of marker genes have not been reported thus far. In this study we confirm that macrophages from common carp can polarize into M1- and M2 phenotypes with conserved functions and corresponding transcriptional profiles compared to mammalian macrophages. Carp M1 macrophages show increased production of nitric oxide and a transcriptional profile with increased pro-inflammatory cytokines and mediators, including il6, il12 and saa. Carp M2 macrophages show increased arginase activity and a transcriptional profile with increased anti-inflammatory mediators, including cyr61, timp2b and tgm2b. Our RNA sequencing approach allowed us to list, in an unbiased manner, markers discriminating between M1 and M2 macrophages of teleost fish. We discuss the importance of our findings for the evaluation of immunostimulants for aquaculture and for the identification of gene targets to generate transgenic zebrafish for detailed studies on M1 and M2 macrophages. Above all, we discuss the striking degree of evolutionary conservation of macrophage polarization in a lower vertebrate.