Comprehensive genome­wide analysis of the chicken heat shock protein family: identification, genomic organization, and expression profiles in indigenous chicken with highly pathogenic avian influenza infection.

BACKGROUND: Heat shock proteins (HSPs) function as molecular chaperones with critical roles in chicken embryogenesis, immune response to infectious diseases, and response to various environmental stresses. However, little is known on HSP genes in chicken. In this study, to understand the roles of chicken HSPs, we performed genome-wide identification, expression, and functional analyses of the HSP family genes in chicken. RESULTS: A total of 76 HSP genes were identified in the chicken genome, which were further classified into eight distinct groups (I-VIII) based on phylogenetic tree analysis. The gene-structure analysis revealed that the members of each clade had the same or similar exon-intron structures. Chromosome mapping suggested that HSP genes were widely dispersed across the chicken genome, except in chromosomes 16, 18, 22, 25, 26, and 28-32, which lacked chicken HSP genes. On the other hand, the interactions among chicken HSPs were limited, indicating that the remaining functions of HSPs could be investigated in chicken. Moreover, KEGG pathway analysis showed that the HSP gene family was involved in the regulation of heat stress, apoptotic, intracellular signaling, and immune response pathways. Finally, RNA sequencing data revealed that, of the 76 chicken HSP genes, 46 were differentially expressed at 21 different growth stages in chicken embryos, and 72 were differentially expressed on post-infection day 3 in two indigenous Ri chicken lines infected with highly pathogenic avian influenza. CONCLUSIONS: This study provides significant insights into the potential functions of HSPs in chicken, including the regulation of apoptosis, heat stress, chaperone activity, intracellular signaling, and immune response to infectious diseases.

An improvement of real-time polymerase chain reaction system based on probe modification is required for accurate detection of African swine fever virus in clinical samples in Vietnam.

OBJECTIVE: The rapid and reliable detection of the African swine fever virus (ASFV) plays an important role in emergency control and preventive measures of ASF. Some methods have been recommended by FAO/OIE to detect ASFV in clinical samples, including realtime polymerase chain reaction (PCR). However, mismatches in primer and probe binding regions may cause a false-negative result. Here, a slight modification in probe sequence has been conducted to improve the qualification of real-time PCR based on World Organization for Animal Health (OIE) protocol for accurate detection of ASFV in field samples in Vietnam. METHODS: Seven positive confirmed samples (four samples have no mismatch, and three samples contained one mutation in probe binding sites) were used to establish novel real-time PCR with slightly modified probe (Y = C or T) in comparison with original probe recommended by OIE. RESULTS: Both real-time PCRs using the OIE-recommended probe and novel modified probe can detect ASFV in clinical samples without mismatch in probe binding site. A high correlation of cycle quantification (Cq) values was observed in which Cq values obtained from both probes arranged from 22 to 25, suggesting that modified probe sequence does not impede the qualification of real-time PCR to detect ASFV in clinical samples. However, the samples with one mutation in probe binding sites were ASFV negative with OIE recommended probe but positive with our modified probe (Cq value ranked between 33.12-35.78). CONCLUSION: We demonstrated for the first time that a mismatch in probe binding regions caused a false negative result by OIE recommended real-time PCR, and a slightly modified probe is required to enhance the sensitivity and obtain an ASF accurate diagnosis in field samples in Vietnam.

Chicken novel leukocyte immunoglobulin-like receptor subfamilies B1 and B3 are transcriptional regulators of major histocompatibility complex class I genes and signaling pathways.

OBJECTIVE: The inhibitory leukocyte immunoglobulin-like receptors (LILRBs) play an important role in innate immunity. The present study represents the first description of the cloning and structural and functional analysis of LILRB1 and LILRB3 isolated from two genetically disparate chicken lines. METHODS: Chicken LILRB1-3 genes were identified by bioinformatics approach. Expression studies were performed by transfection, quantitative polymerase chain reaction. Signal transduction was analyzed by western blots, immunoprecipitation and flow cytometric. Cytokine levels were determined by enzyme-linked immunosorbent assay. RESULTS: Amino acid homology and phylogenetic analyses showed that the homologies of LILRB1 and LILRB3 in the chicken line 6.3 to those proteins in the chicken line 7.2 ranged between 97%-99%, while homologies between chicken and mammal proteins ranged between 13%-19%, and 13%-69%, respectively. Our findings indicate that LILRB1 and LILRB3 subdivided into two groups based on the immunoreceptor tyrosine-based inhibitory motifs (ITIM) present in the transmembrane domain. Chicken line 6.3 has two ITIM motifs of the sequence LxYxxL and SxYxxV while line 7.2 has two ITIM motifs of the sequences LxYxxL and LxYxxV. These motifs bind to SHP-2 (protein tyrosine phosphatase, non-receptor type 11) that plays a regulatory role in immune functions. Moreover, our data indicate that LILRB1 and LILRB3 associated with and activated major histocompatibility complex (MHC) class I and ß2-microglobulin and induced the expression of transporters associated with antigen processing, which are essential for MHC class I antigen presentation. This suggests that LILRB1 and LILRB3 are transcriptional regulators, modulating the expression of components in the MHC class I pathway and thereby regulating immune responses. Furthermore, LILRB1 and LILRB3 activated Janus kinase2/tyrosine kinase 2 (JAK2/TYK2); signal transducer and activator of transcription1/3 (STAT1/3), and suppressor of cytokine signaling 1 genes expressed in Macrophage (HD11) cells, which induced Th1, Th2, and Th17 cytokines. CONCLUSION: These data indicate that LILRB1 and LILRB3 are innate immune receptors associated with SHP-2, MHC class I, ß2-microglobulin, and they activate the Janus kinase/signal transducer and activator of transcription signaling pathway. Thus, our study provides novel insights into the regulation of immunity and immunopathology.

Leukocyte Immunoglobulin-Like Receptors A2 and A6 are Expressed in Avian Macrophages and Modulate Cytokine Production by Activating Multiple Signaling Pathways.

The activating leukocyte immunoglobulin-like receptors (LILRAs) play an important role in innate immunity. However, most of the LILRA members have not been characterized in avian species including chickens. The present study is the first attempt at cloning, structural analysis and functional characterization of two LILRAs (LILRA2 and LILRA6) in chickens. Multiple sequence alignments and construction of a phylogenetic tree of chicken LILRA2 and LILRA6 with mammalian proteins revealed high conservation between chicken LILRA2 and LILRA6 and a close relationship between the chicken and mammalian proteins. The mRNA expression of LILRA2 and LILRA6 was high in chicken HD11 macrophages and the small intestine compared to that in several other tissues and cells tested. To examine the function of LILRA2 and LILRA6 in chicken immunity, LILRA2 and LILRA6 were transfected into HD11 cells. Our findings indicated that LILRA2 and LILRA6 are associated with the phosphorylation of Src kinases and SHP2, which play a regulatory role in immune functions. Moreover, LILRA6 associated with and activated MHC class I, ß2-microglobulin and induced the expression of transporters associated with antigen processing but LILRA2 did not. Furthermore, both LILRA2 and LILRA6 activated JAK-STAT, NF-κB, PI3K/AKT and ERK1/2 MAPK signaling pathways and induced Th1-, Th2- and Th17-type cytokines and Toll-like receptors. Collectively, this study indicates that LILRA2 and LILRA6 are essential for macrophage-mediated immune responses and they have the potential to complement the innate and adaptive immune system against pathogens.

Validation of a direct multiplex real-time reverse transcription PCR assay for rapid detection of African swine fever virus using swine field samples in Vietnam.

OBJECTIVE: This study validates a direct multiplex real-time reverse transcription polymerase chain reaction (rRT-PCR) assay which was previously established for enabling rapid and simultaneous detection of African swine fever (ASF) virus (ASFV) and classical swine fever virus. The assay eliminates the need for viral nucleic acid purification using a buffer system for crude extraction and an impurity-tolerant enzyme. However, the assay had not yet been validated using field samples of ASFV-infected pigs. Therefore, to address this gap, we tested 101 samples collected from pigs in Vietnam during 2018 and 2021 for validation. RESULTS: The rRT-PCR assay demonstrated a diagnostic sensitivity of 98.8% and a specificity of 100%. Remarkably, crude samples yielded results comparable to those of purified samples, indicating the feasibility of using crude samples without compromising accuracy in ASFV detection. Our findings emphasize the effectiveness of the rRT-PCR assay for the prompt and accurate diagnosis of both swine fever viruses, which is essential for effective disease prevention and control in swine populations.

A new variant of lumpy skin disease virus circulating in Vietnam based on sequencing analysis of GPCR gene.

Background: In 2021, Vietnam experienced an outbreak of Lumpy skin disease (LSD), which infected 207,687 cattle and buffaloes, as officially reported, and resulted in the culling of 29,182 animals. Aim: In this study, samples from cattle that died and showed typical signs of LSD in the Ha Tinh province of Vietnam were confirmed by three World Organization for Animal Health (WOAH)-recommended methods and further studied to compare the Vietnam and China reference strains to the new clinical cases. Methods: Three methods recommended by WOAH for agent detection (PCR, virus isolation, and transmission electron microscopy) were used to confirm this clinical LSD case. The sequence analysis of three well-known markers (P32, RPO30, and GPCR genes) has been utilized in Vietnam to understand this circulating pathogen better. Results: Our findings showed that the CX01 LSDV strain is 100% identical to the Vietnam reference strain HL01 and China reference strains based on P32 and RPO30 genes. Interestingly, analysis of the nucleotide sequence of the GPCR gene showed that the CX01 strain belongs to the same cluster as the reference strains, but it has branches different from those of both the HL01 and China LSDV strains. The nucleotide identification between the CX01 strain and these reference virus strains ranked 99.65%-99.91%, suggesting that it is a new variant of LSDV. Conclusion: This finding is new and indicates that at least two variants of the LSD virus were circulating in Vietnam based on analysis of the GPCR gene. Additionally, these results suggest that the sequence analysis of the GPCR gene is a great tool for subgrouping LSDV circulating in Vietnam.

Identification of immune-associated genes with altered expression in the spleen of mice enriched with probiotic Lactobacillus species using rna-seq profiling.

Objective: Probiotics are living microorganisms that can provide health benefits when consumed. Here, we investigated the effects of probiotics on gene expression in the spleen of mice using RNA-sequencing analysis between negative control and probiotic groups (including 4 Lactobacillus strains: Lactobacillus fermentum, L. casei, L. plantarum, and L. brevis). Methods: Mice exposed with probiotic in 4 weeks by intragastric administration. Then, spleen tissues of the control and probiotics groups were collected on days 14 and 28 for RNA sequencing. Results: In total, 665, 186, and 81 DEGs were significantly expressed on day 14 vs. control, day 28 vs. control groups, and probiotics day 28 vs. day 14 groups, respectively. On the other hand, 12 Toll-like receptor (TLR) genes underwent additional validation through qRT-PCR, affirming the increased alignment between qRT-PCR and RNA-Seq findings. In addition, the Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) analyses revealed that the DEGs were predominantly enriched in defense responses to pathogens, including inflammatory bowel diseases, malaria, leukaemia virus 1, and herpes virus, as well as immune processes related to immune response and signal transduction. This study represents the first investigation into mice's gene expression in the spleen exposed to probiotics using Lactobacillus spp. isolated from a field strain in Vietnam. Conclusion: Our results provide valuable insights into the impacts and functions of probiotics on mammalian development, offering crucial information for the potential therapeutic use of probiotics in defending against pathogens in Vietnam. The findings from this study highlight the potential of probiotics in modulating gene expression in the spleen, which may have implications for immune function and overall health in mice.

Molecular characterization of lumpy skin disease virus in North Central Vietnam during 2021 and early 2022.

In October 2020, the first outbreaks of lumpy skin disease (LSD) in Lang Son Province, Vietnam were reported by our laboratory. The disease had rapidly spread to the South, and it was reported in 55 of 63 provinces and cities of Vietnam by the end of 2021. The most economic loss caused by this disease occurred in the north-central region in 2021 where approximately 46,788 LSD virus (LSDV) infected cattle and buffaloes have been reported and 8,976 animals have been culled. However, the information on this pathogen circulating in this region is missing. Here, we describe the molecular characterization of LSDV circulating in north-central Vietnam in 2021 and early 2022. In total, 155 LSDV samples were collected during this period and three of these samples from each province were further characterized by Sanger sequencing analysis based on three key maker genes (GPCR, RPO30, and p32). Sequence comparison and phylogenetic analysis based on GPCR, RPO30, and p32 genes indicated that LSDV strains circulating in north-central Vietnam are closely related to previously reported strains in Vietnam regions which bordered China and all LSDV strains were 100% identical. These results show the importance of continuous monitoring and characterization of circulating LSDV strains and are important for vaccine development for the control and eradication of LSD in Vietnam.

Effect of saponins in Panax notoginseng (Burkill) F. H. Chen on the steroid hormone levels in the chronic unpredictable mild stress model of depression in rats.

Recent research has indicated that Panax notoginseng saponins (PNS) extracted from the radix of Panax notoginseng (Burkill) F. H. Chen exert antidepressant effects. This study aimed to assess the antidepressive effects of ginsenoside Rg1 and PNS in a depression model induced by chronic unpredictable mild stress (CUMS). Over a period of three weeks, rats were administered ginsenoside Rg1 at a dose of 30 mg/kg and PNS at dosages ranging from 100 to 200 mg/kg body weight per day. To assess how ginsenoside Rg1 and PNS influence depression-like behaviours in rats, various assessments were conducted, including coat state evaluation, forced swim test, and elevated plus maze test. The levels of cortisol and testosterone in serum samples were analysed using the liquid chromatography-electrospray ionisation tandem mass spectrometry (LC-ESI-MS/MS) method. LC-ESI-MS/MS method provides precise and accurate results. The lower limit of quantification values for cortisol and testosterone were determined as 100 and 2 pg/mL, respectively. Our data demonstrated that both ginsenoside Rg1 and PNS significantly reversed depression-like behaviour in rats by improving coat condition, reducing immobility time in the forced swim test, and increasing time spent in the open arms of the elevated plus maze test. Furthermore, ginsenoside Rg1 and PNS exhibited a regulatory effect on cortisol and testosterone levels in plasma. These findings suggest that ginsenoside Rg1 and PNS may be potential antidepressants in clinical treatment.

Molecular analysis of chicken interferon-alpha inducible protein 6 gene and transcriptional regulation.

Interferon-alpha inducible protein 6 (IFI6) is an interferon-stimulated gene (ISG), belonging to the FAM14 family of proteins and is localized in the mitochondrial membrane, where it plays a role in apoptosis. Transcriptional regulation of this gene is poorly understood in the context of inflammation by intracellular nucleic acid-sensing receptors and pathological conditions caused by viral infection. In this study, chicken IFI6 (chIFI6) was identified and studied for its molecular features and transcriptional regulation in chicken cells and tissues, i.e., lungs, spleens, and tracheas from highly pathogenic avian influenza virus (HPAIV)-infected chickens. The chIFI6-coding sequences contained 1638 nucleotides encoding 107 amino acids in three exons, whereas the duck IFI6-coding sequences contained 495 nucleotides encoding 107 amino acids. IFI6 proteins from chickens, ducks, and quail contain an IF6/IF27-like superfamily domain. Expression of chIFI6 was higher in HPAIV-infected White Leghorn chicken lungs, spleens, and tracheas than in mock-infected controls. TLR3 signals regulate the transcription of chIFI6 in chicken DF-1 cells via the NF-κB and JNK signaling pathways, indicating that multiple signaling pathways differentially contribute to the transcription of chIFI6. Further research is needed to unravel the molecular mechanisms underlying IFI6 transcription, as well as the involvement of chIFI6 in the pathogenesis of HPAIV in chickens.

Biological properties and diverse cytokine profiles followed by in vitro and in vivo infections with LSDV strain isolated in first outbreaks in Vietnam.

Preliminary information about LSD virus isolated from the first outbreaks in Vietnam has been reported by our laboratory. In the current study, LSDV strain, LSDV/Vietnam/Langson/HL01(HL01) was further analyzed to provide a better understanding of this viral pathogen. HL01 LSDV strain was propagated at MOI 0.01 in MDBK cells and then given to cattle at dose of 106.5 TCID50/ml (2ml/animal). The production of proinflammatory (IFN-γ, IL-1α, and TNF-α) and anti-inflammatory (IL-6, IL-10, and TGF-ß1) cytokines were measured by real-time PCR, both In vitro and In vivo. The results demonstrated that HL01 strain caused the typical signs of LSD and LSDV In vitro and In vivo, respectively suggesting a virulent field LSDV strain. Additionally, different cytokine profiles were observed in these In vitro and In vivo studies. In MDBK cells, different cytokines profiles were observed in two phases: in the early phase, the expression levels of all examined cytokines were significantly increased at 6 h (p < 0.05). In the later phase, the peak levels of the cytokine secretion were recognized from 72 to 96 h, with the exception of IL-1α when compared to controls. In cattle, the expression levels of all six cytokines were significantly higher at day 7 following LSDV challenge (p < 0.05) when compared to controls, especially expression levels of TGF-ß1 and IL-10. These findings suggest the important roles of these cytokines in protection against LSDV infections. Additionally, the data from diverse cytokine profiles followed by this LSDV strain challenge provides key understanding of the underlying cellular immune mechanisms in the host against LSDV infection In vitro and In vivo.

HPAI-resistant Ri chickens exhibit elevated antiviral immune-related gene expression.

BACKGROUND: Highly pathogenic avian influenza viruses (HPAIVs) is an extremely contagious and high mortality rates in chickens resulting in substantial economic impact on the poultry sector. Therefore, it is necessary to elucidate the pathogenic mechanism of HPAIV for infection control. OBJECTIVE: Gene set enrichment analysis (GSEA) can effectively avoid the limitations of subjective screening for differential gene expression. Therefore, we performed GSEA to compare HPAI-infected resistant and susceptible Ri chicken lines. METHODS: The Ri chickens Mx(A)/BF2(B21) were chosen as resistant, and the chickens Mx(G)/BF2(B13) were selected as susceptible by genotyping the Mx and BF2 genes. The tracheal tissues of HPAIV H5N1 infected chickens were collected for RNA sequencing followed by GSEA analysis to define gene subsets to elucidate the sequencing results. RESULTS: We identified four differentially expressed pathways, which were immune-related pathways with a total of 78 genes. The expression levels of cytokines (IL-1ß, IL-6, IL-12), chemokines (CCL4 and CCL5), type interferons and their receptors (IFN-ß, IFNAR1, IFNAR2, and IFNGR1), Jak-STAT signaling pathway genes (STAT1, STAT2, and JAK1), MHC class I and II and their co-stimulatory molecules (CD80, CD86, CD40, DMB2, BLB2, and B2M), and interferon stimulated genes (EIF2AK2 and EIF2AK1) in resistant chickens were higher than those in susceptible chickens. CONCLUSIONS: Resistant Ri chickens exhibit a stronger antiviral response to HPAIV H5N1 compared with susceptible chickens. Our findings provide insights into the immune responses of genetically disparate chickens against HPAIV.

Genome­wide identification, organization, and expression profiles of the chicken fibroblast growth factor genes in public databases and Vietnamese indigenous Ri chickens against highly pathogenic avian influenza H5N1 virus infection.

OBJECTIVE: Fibroblast growth factors (FGFs) play critical roles in embryo development, and immune responses to infectious diseases. In this study, to investigate the roles of FGFs, we performed genome-wide identification, expression, and functional analyses of FGF family members in chickens. METHODS: Chicken FGFs genes were identified and analyzed by using bioinformatics approach. Expression profiles and Hierarchical cluster analysis of the FGFs genes in different chicken tissues were obtained from the genome-wide RNA-seq. RESULTS: A total of 20 FGF genes were identified in the chicken genome, which were classified into seven distinct groups (A-F) in the phylogenetic tree. Gene structure analysis revealed that members of the same clade had the same or similar exon-intron structure. Chromosome mapping suggested that FGF genes were widely dispersed across the chicken genome and were located on chromosomes 1, 4-6, 9-10, 13, 15, 28, and Z. In addition, the interactions among FGF proteins and between FGFs and mitogen­activated protein kinase (MAPK) proteins are limited, indicating that the remaining functions of FGF proteins should be further investigated in chickens. Kyoto encyclopedia of genes and genomes pathway analysis showed that FGF gene interacts with MAPK genes and are involved in stimulating signaling pathway and regulating immune responses. Furthermore, this study identified 15 differentially expressed genes (DEG) in 21 different growth stages during early chicken embryo development. RNA-sequencing data identified the DEG of FGFs on 1- and 3-days post infection in two indigenous Ri chicken lines infected with the highly pathogenic avian influenza virus H5N1 (HPAIV). Finally, all the genes examined through quantitative real-time polymerase chain reaction and RNA-Seq analyses showed similar responses to HPAIV infection in indigenous Ri chicken lines (R2 = 0.92- 0.95, p<0.01). CONCLUSION: This study provides significant insights into the potential functions of FGFs in chickens, including the regulation of MAPK signaling pathways and the immune response of chickens to HPAIV infections.

Molecular and functional characterization of chicken interleukin 1 receptor 2 (chIL-1R2).

Interleukin-1 receptor type 2 (IL1R2) is a decoy receptor for exogenous IL-1. However, its functional role in chicken immunity is poorly understood. Herein, chicken IL-1R2 (chIL-1R2) was identified and functionally characterized in vivo and in vitro. The chIL-1R2 coding sequence includes 1,236 nucleotides encoding 412 amino acids, is highly conserved, and has a close relationship with its mammalian counterpart. Its extracellular region has three Ig-like domains but no TIR domain for intracellular signaling. Using ELISA, the recombinant chIL-1R2 protein was demonstrated to specifically bind to the chicken IL-1ß. ChIL-1R2 mRNA expression was shown to be higher in the spleen, lung, kidney, small intestine, and liver. The expression of chIL-1R2 and chIL-1R1 was significantly upregulated in DF-1 cells treated with poly (I:C), but significantly downregulated in the presence of NF-κB, JNK, and MEK inhibitors, indicating that the NF-κB, JNK, and MEK signaling pathways are required for the transcriptional regulation of chIL-1R1 and chIL-1R2 expression. It is worth noting that while the p30 MAPK pathway was required for chIL-1R1 expression, it was not required for chIL-1R2 expression. Furthermore, chIL-1R2 expression increased as early as day 1, and then significantly decreased until day 3, while chIL-1R1 was dramatically upregulated in four organs of chickens infected with the highly pathogenic avian influenza virus (HPAIV). These findings indicate that chIL-1R1 and chIL-1R2 may play a crucial in innate and adaptive immune responses toward HPAIV infection. In summary the present study showed that chIL-1R2 binds to chIL-1ß antibody. ChIL-1R2 expression can be induced by a viral infection, and may be regulated through NF-κB/JNK/MEK-mediated signaling pathways.

Small RNA sequencing and profiling of serum-derived exosomes from African swine fever virus-infected pigs.

African swine fever (ASF) virus (ASFV) is responsible for one of the most severe swine diseases worldwide, with a morbidity rate of up to 100%; no vaccines or antiviral medicines are available against the virus. Exosomal miRNAs from individual cells can regulate the immune response to infectious diseases. In this study, pigs were infected with an ASFV Pig/HN/07 strain that was classified as acute form, and exosomal miRNA expression in the serum of infected pigs was analyzed using small RNA sequencing (small RNA-seq). Twenty-seven differentially expressed (DE) miRNAs were identified in the ASFV-infected pigs compared to that in the uninfected controls. Of these, 10 were upregulated and 17 were downregulated in the infected pigs. All DE miRNAs were analyzed using gene ontology (GO) terms and the Kyoto Encyclopedia of Genes and Genomes (KEGG) database, and the DE miRNAs were found to be highly involved in T-cell receptor signaling, cGMP-PKG signaling, Toll-like receptor, MAPK signaling, and mTOR signaling pathways. Furthermore, the Cytoscape network analysis identified the network of interactions between DE miRNAs and target genes. Finally, the transcription levels of four miRNA genes (ssc-miR-24-3p, ssc-miR-130b-3p, ssc-let-7a, and ssc-let-7c) were examined using quantitative real-time PCR (qRT-PCR) and were found to be consistent with the small RNA-seq data. These DE miRNAs were associated with cellular genes involved in the pathways related to immune response, virus-host interactions, and several viral genes. Overall, our findings provide an important reference and improve our understanding of ASF pathogenesis and the immune or protective responses during an acute infection in the host.

African swine fever is a viral disease caused by African swine fever virus (ASFV) which induces a big threat to the pig industry in the world. To date, there are no vaccines or antiviral medicines against the ASFV. Therefore, it is important to improve the understanding of the pathogenesis of ASFV and host­pathogen interaction using miRNA that may regulate genes related to the immune system. This study aimed to investigate the differentially expressed (DE) miRNA in serum-derived exosomes from African swine fever virus infected pigs. We successfully infected pigs with an ASFV Pig/HN/07 strain and identified the DE miRNAs in serum-derived exosomes using small RNA sequencing. Our results showed that total of 27 miRNAs were differentially expressed in serum-derived exosomes from ASFV-infected pigs. We analyzed the small RNA sequencing results using gene ontology (GO) terms and the Kyoto Encyclopedia of Genes and Genomes (KEGG) database and found that most DE miRNA may regulate the expression of genes related with the immune response pathway (T-cell receptor signaling pathway, cGMP-PKG signaling pathway, PI3K-Akt signaling pathway, MAPK signaling pathway, etc.).

Whole Genome Sequencing of African Swine Fever.

Next-generation sequencing (NGS) technologies have been powerfully applied in both research and clinical settings for the understanding and control of infectious disease. It enables high-resolution characterization of viral pathogens in terms of properties that include molecular epidemiology, genotype, serotype, and virulence. However, a beginner's NGS protocol for characterization of African swine fever virus (ASFV) is lacking. Here, we present detailed step-by-step methods for obtaining NGS data from ASF virus (ASFV) using the Illumina platform. The protocol has been performed with respect to ASFV DNA genome extraction, qualification of DNA, library preparation, quality control, de novo assembly, and data quality control. The protocol represents a step-by-step and reproducible method for producing high-quality sequencing data. The key advantages of this protocol include the protocol being very simple for users with no experience of genome sequencing and reproducibility of the protocol for other DNA genome viruses.

Application of Fourier transform-based algorithms to resolve spectral overlapping for UV spectrophotometric co-assay of spiramycin and metronidazole in tablets.

Fourier transform-based algorithms were investigated to resolve UV spectral overlapping of spiramycin and metronidazole in binary mixtures. UV spectra and ratio spectra were both subjected to fast Fourier transform-based first-order differentiation and discrete Fourier transform {i.e. using trigonometric functions such as sin xi - sin (xi + 450), cos xi + cos (xi + 450), sin 2xi - sin 2(xi + 450), cos 2xi + cos 2(xi + 450), sin xi - sin (xi + 600), cos xi + cos (xi + 600)} that followed by 3 passes of 17-point triangular smooth. Such signal transforms generated linear calibration graphs for either drug in the concentration range of 6.25 - 25 mg/L with R2 > 0.990. In comparison with the RP-HPLC reference method, the developed UV spectrophotometric methods had no significant difference in terms of accuracy and precision (p > 0.05) when quantifying spiramycin and metronidazole in their coated tablets. They are suggested as analytical quality control methods for their being environmentally friendly, technically simple, quick and economic.

Inhibition of African swine fever virus replication by ß-glucan.

Background: African swine fever (ASF) is one of the most important diseases in pigs because of its effects on all ages and breeds. To date, commercial vaccines and drugs for the prevention of ASF are lacking in the market and the survival of African swine fever virus (ASFV) in various environmental, farm, and or feed matrices has allowed the virus to remain, causing new outbreaks in the pig population. Besides biosecurity and animal husbandry management practices, the improvement of the host immune responses is critical to control, managing, and preventing ASF. Aim: In this study, we investigated the protective role of ß-glucan against ASFV infection using a porcine alveolar macrophage (PAM) model. Methods: The effects of ß-glucan on cell proliferation were evaluated by using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. The potential effects of ß-glucan against a field ASFV strain isolated in Vietnam were further examined by real-time PCR and hemadsorption assays. The interferon (IFN)-α and interleukin (IL)-6 protein production induced by ß-glucan was determined using a sandwich enzyme-linked immunosorbent assay. Results: Our results demonstrated that the ß-glucan additive possessed an immune stimulus factor against ASFV. Specifically, protection of PAMs against ASFV infection in vitro was observed at 12 hours (p < 0.05) at the tested doses (30 and 50 µg/ml) as induced by incubation with ß-glucan for 2 hours. These effects remained until 24 hours after post-infection. Additionally, at a high dose (50 µg/ml), pre-treatment with the ß-glucan statistically increased the expression levels of IFNα and IL-6 when compared to untreated groups or only ASFV infection. Conclusion: Together, these findings indicated that the ß-glucan may protect the host against ASFV infection via the multiple cellular immune mechanisms.

Cytokine-cytokine receptor interactions in the highly pathogenic avian influenza H5N1 virus-infected lungs of genetically disparate Ri chicken lines.

OBJECTIVE: The highly pathogenic avian influenza virus (HPAIV) is a threat to the poultry industry as well as the economy and remains a potential source of pandemic infection in humans. Antiviral genes are considered a potential factor for HPAIV resistance. Therefore, in this study, we investigated gene expression related to cytokine-cytokine receptor interactions by comparing resistant and susceptible Ri chicken lines for avian influenza virus infection. METHODS: Ri chickens of resistant (Mx/A; BF2/B21) and susceptible (Mx/G; BF2/B13) lines were selected by genotyping the Mx dynamin like GTPase (Mx) and major histocompatibility complex class I antigen BF2 genes. These chickens were then infected with influenza A virus subtype H5N1, and their lung tissues were collected for RNA sequencing. RESULTS: In total, 972 differentially expressed genes (DEGs) were observed between resistant and susceptible Ri chickens, according to the gene ontology and Kyoto encyclopedia of genes and genomes pathways. In particular, DEGs associated with cytokine-cytokine receptor interactions were most abundant. The expression levels of cytokines (interleukin-1ß [IL-1ß], IL-6, IL-8, and IL-18), chemokines (C-C Motif chemokine ligand 4 [CCL4] and CCL17), interferons (IFN-γ), and IFN-stimulated genes (Mx1, CCL19, 2'-5'-oligoadenylate synthaselike, and protein kinase R) were higher in H5N1-resistant chickens than in H5N1-susceptible chickens. CONCLUSION: Resistant chickens show stronger immune responses and antiviral activity (cytokines, chemokines, and IFN-stimulated genes) than those of susceptible chickens against HPAIV infection.

Novel method for sub-grouping of genotype II African swine fever viruses based on the intergenic region between the A179L and A137R genes.

BACKGROUND: African swine fever (ASF) is a highly contagious and deadly viral disease affecting domestic and wild pigs of all ages. African swine fever virus (ASFV) has spread rapidly through Eastern and Southeastern Asia first appearing in Vietnam in 2019. OBJECTIVES: Molecular typing of African swine fever virus (ASFV) in Vietnam has identified two principal variants circulating based on the sequencing of the intergenic region (IRG) between the I73R and I329L genes. Identification of additional genetic markers would enable higher resolution tracing of outbreaks within the country. METHODS: Sequence analysis suggested the IRG between the A179L and A137R genes may also exhibit variability, PCR primers were designed and samples from Vietnam were subject to Sanger sequencing. RESULTS: We developed a novel method for sub-grouping of ASFV based on the IRG between the A179L and A137R genes of ASFV. Our results demonstrated that the finding of the insertion or deletion of an 11- nucleotide sequence (GATACAATTGT) between the A179L-A137R genes. CONCLUSIONS: The sub-grouping method may provide useful insights into the evolution of genotype II ASFV as well as providing evidence of a relationship between geographically separated outbreaks.