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.

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.

Bioinformatic identification and expression analysis of the chicken B cell lymphoma (BCL) gene.

BACKGROUND: B cell lymphoma (BCL) families play an important role in apoptosis as a growth factor, cell death programming, cytokine expression and immune-related genes expression. OBJECTIVES: In this study, to investigate the roles of BCLs, we performed genome-wide identification, expression and functional analyses of the BCL family in chicken. METHODS: Chicken BCLs genes were identified and analyzed by using bioinformatics approach. Expression profiles and Hierarchical cluster analysis of the BCLs genes in different chicken tissues were obtained from the genome-wide RNA-seq in the GEO, and Cluster and Java Treeview, respectively. RESULTS: A total of 16 BCLs genes were identified from the chicken genome, which could be further classified into five distinct groups in the phylogenetic tree. On the other hand, the interaction among BCLs proteins and between BCLs proteins with NF-κB subunits are limited, indicating that the remaining the functions of BCLs protein could be investigated in chicken. Moreover, KEGG pathway analysis indicated that BCL gene family was involved in regulation of apoptotic and immune response. Finally, BCL gene family was differentially expressed in chicken tissues, pathogen infection and growth stages of early chicken early embryo. CONCLUSION: This study provides significant insights into the potential functions of BCLs in chicken, including the regulation of apoptosis, cell death and expression of immune-related genes.