Identification of differentially expressed genes and metabolism signaling pathway in the spleen of broilers supplemented with probiotic Bacillus spp.

Probiotics are essential in the body's nutrients, improving the ratio of meat to meat, immune response, and preventing diseases. In this study, RNA-sequencing (RNA-seq) was used to identify the differentially expressed genes (DEGs), enriched related pathways, and Gene Ontology (GO) terms among blank negative control (NC), supplemented with Bacillus spp. (BS) and commercial probiotic (PC) groups after a 42-day fed supplementation. The results showed that 2005, 1356, and 2189 DEGs were significantly altered in BS vs. NC, PC vs NC, and BS vs PC groups, respectively. On the other hand, 9 DEGs were further validated by qRT-PCR, indicating that the qRT-PCR and RNA-Seq results were more consistent. Therefore, the GO and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses of DEGs showed that the DEGs were mainly enriched to metabolism signalling pathways (alpha-linolenic acid metabolism, linoleic acid metabolism, tryptophan metabolism, tyrosine metabolism, ether lipid metabolism, and metabolic pathway, etc) and immune response pathways (cytokine-cytokine receptor interaction, MAPK signalling pathway, and intestinal immune network for IgA production, neuroactive ligand-receptor interaction etc). These results will provide a better understanding of the role of probiotics in chicken development and provide basic information on the genetic development of chickens.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

The potential anti-African swine fever virus effects of medium chain fatty acids on in vitro feed model: An evaluation study using epidemic ASFV strain circulating in Vietnam.

Background: African swine fever (ASF) is an important disease affecting swine and has a significant economic loss in both the developed and developing world. Aim: In this study, we evaluated the potential effects of medium-chain fatty acids (MCFAs) in individual and synergistic forms to prevent and/or reduce ASF virus (ASFV) infection using in vitro feed model. Methods: The cytotoxicity of MCFAs on porcine alveolar macrophages cells was evaluated by using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. The potential effects of MCFAs, including C8 (caprylic acid), C8-C6-C10 (caprylic acid-caproic acid-capric acid; 1:1:1 ratio) and C8-C10-C12 (caprylic acid-capric acid-lauric acid; 1:1:1 ratio) against a field ASFV strain isolated in the capital Hanoi of Vietnam, were further examined by real-time PCR and haemadsorption assays in in vitro feed model. Results: Our results indicated that all tested products do not induce cytotoxicity at the dose of 100 µg/ml and are suitable for further in vitro examination. These products have shown a strong antiviral effect against ASFV infectivity at doses of 0.375% and 0.5%. Interestingly, the synergistic MCFAs have shown clearly their potential activities against ASFV in which at a lower dose of 0.25%, pre-treatment with product two and three induced significant increases at the level of Cq value when compared to positive control and/or product 1 (p < 0.05). However, the viral titre was not changed after 24 hours post-inoculation when compared to positive control. Our findings suggested that all tested products, both individual and synergistic forms of MCFAs, have possessed a strong anti-ASFV effect, and this effect is dose-dependence in in vitro feed model. Additionally, synergistic effects of MCFAs are more effective against ASFV when compared to individual forms. Conclusion: Together, the findings in this study indicate that MCFAs, both individual and synergistic forms, inhibit against a field ASFV strain in the feed model, which may support minimizing the risk of ASF transmission in the pig population. Further studies focusing on in vivo anti-ASFV effects of MCFAs are important to bring new insight into the mode of ASFV-reduced action by these compounds in swine feed.

Lumpy skin disease outbreaks in vietnam, 2020.

Lumpy skin disease (LSD) is a transboundary, systemic, viral disease of cattle. The first outbreaks of LSD were reported in Lang Son Province of Vietnam (bordered to China), and an official document has been submitted to OIE on 1 November 2020. Here, we described first the genetic profiles of this pathogen based on four well-known marker regions. The LSD virus isolated in these first outbreaks was 100% identical to viruses isolated in China (2019) based on the p32 and RP030 genes. Additionally, it is very close to the virus isolated in Russia (2017) based on the p32, RP030, thymidine kinase and ORF103 genes (100%, 99.01%, 99.08% and 99.47% identities). This finding is new, and a success in LSD virus isolation using MDBK cells from first outbreaks is important for vaccine development to control and eradicate LSD in Vietnam.

Circulation of two different variants of intergenic region (IGR) located between the I73R and I329L genes of African swine fever virus strains in Vietnam.

Since African swine fever virus (ASFV) introduction into Vietnam in 2019, most ASFV strains detected in this country belong to the p72 genotype II and intergenic region (IGR) II variant. Further investigation of the intergenic region of ASFVs isolated in the Capital Hanoi region showed two different variants, IGR I and IGR II, which were located between the I73R and I329L genes of the p72 genotype II ASFV strains. This finding suggests co-circulation of two ASFV variants in the domestic pig population in Vietnam.

Genetic characterization of African swine fever viruses circulating in North Central region of Vietnam.

Since the first outbreak of African swine fever virus (ASFV) in China in 2018, the disease has spread to Mongolia, Vietnam, Cambodia, Korea, Laos, Myanmar, Philippines, Timor-Leste, Indonesia and Papua New Guinea. ASFV was officially reported in Vietnam on 19 February 2019. The continued spread of ASFV has occurred in the whole country within 7 months. The phylogenetic analysis showed that ASFVs isolated in the North Central region of Vietnam belong to genotype II and serotype 8. Additionally, tandem repeat sequence (TRS) studies indicated that these ASFVs are very close to ASFV strains detected in China and Belgium, 2018, and differ from ASFV isolated in Georgia in 2007.

Unexpected cases in field diagnosis of African swine fever virus in Vietnam: The needs consideration when performing molecular diagnostic tests.

Background: The first confirmed case of African swine fever (ASF) in Vietnam was reported officially in February 2019. To date, ASF virus (ASFV) have been detected in 63/63 provinces in Vietnam. Currently, real-time polymerase chain reaction (PCR) is considered to be a powerful tool for viral detection in field samples, including ASFV. However, some recent reports have suggested that mismatches in primer and probe binding regions may directly affect real-time PCR qualification, leading a false-negative result. Aim: This study aims to further examine a conflicting result obtained from two OIE recommended methods, conventional PCR and real-time PCR, for ASFV detection. Methods: Two ASF suspected pigs from different provinces in the north of Vietnam were selected for this study based on clinical signs and postmortem lesions. The different results obtained by OIE-recommended conventional PCR and real-time PCR were further analyzed by the Sanger sequencing method and virus isolation in combination with hemadsorption (HAD) test using porcine alveolar macrophages cells. Results: The results showed that when the primer sequence matched perfectly with the sequences of field isolates, a mutation in probe binding region was found, indicating that a single mismatch in the probe binding site may cause a false-negative result by real-time PCR in detecting ASFV in clinical samples in Vietnam. An agreement between conventional PCR, using PPA1/PPA2 primers and two golden standard methods, virus isolation in combination with HAD assay, and sequencing method was observed in this study. Conclusion: A single mismatch in the probe binding site caused a failse-negative result by realtime PCR method in field diagnosis of ASFV. The needs consideration when selecting the appropriate molecular diagnostic methods is based on the current databases of ASFV sequences, particularly for epidemiological surveillance of ASF.

Genetic Characterisation of African Swine Fever Virus in Outbreaks in Ha Nam Province, Red River Delta Region of Vietnam, and Activity of Antimicrobial Products Against Virus Infection in Contaminated Feed.

INTRODUCTION: African swine fever (ASF) was officially reported in Vietnam in February 2019 and spread across the whole country, affecting all 63 provinces and cities. MATERIAL AND METHODS: In this study, ASF virus (ASFV) VN/Pig/HaNam/2019 (VN/Pig/HN/19) strain was isolated in primary porcine alveolar macrophage (PAM) cells from a sample originating from an outbreak farm in Vietnam's Red River Delta region. The isolate was characterised using the haemadsorption (HAD) test, real-time PCR, and sequencing. The activity of antimicrobial feed products was evaluated via a contaminated ASFV feed assay. RESULTS: Phylogenetic analysis of the viral p72 and EP402R genes placed VN/Pig/HN/19 in genotype II and serogroup 8 and related it closely to Eastern European and Chinese strains. Infectious titres of the virus propagated in primary PAMs were 106 HAD50/ml. Our study reports the activity against ASFV VN/Pig/HN/19 strain of antimicrobial Sal CURB RM E Liquid, F2 Dry and K2 Liquid. Our feed assay findings suggest that the antimicrobial RM E Liquid has a strong effect against ASFV replication. These results suggest that among the Sal CURB products, the antimicrobial RM E Liquid may have the most potential as a mitigant feed additive for ASFV infection. Therefore, further studies on the use of antimicrobial Sal CURB RM E Liquid in vivo are required. CONCLUSIONS: Our study demonstrates the threat of ASFV and emphasises the need to control and eradicate it in Vietnam by multiple measures.

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.