The Viremic Phase and Humoral Immune Response Against African Horse Sickness Virus That Emerged in Thailand in 2020
Simple Summary
Abstract
1. Introduction
2. Materials and Methods
2.1. Study Design and Sample Collection
2.2. RNA Isolation and cDNA Synthesis
2.3. AHSV Detection Using Group-Specific (VP7) RT-PCR and RT Real-Time PCR
2.4. VP5 Gene Sequencing and Phylogenetic Analysis
2.5. Type-Specific (VP5) RT Nested PCR and RT Real-Time PCR for AHSV-1 Thai Field Strain and Vaccine Strain Differentiation
2.6. Immune Response Against AHSV by Blocking ELISA (bELISA)
2.7. Statistics
3. Results
3.1. Characterization and Phylogenetic Analysis of VP5 Gene
3.2. Viremic Phase and Clinical Signs of Naturally Infected Naïve Horses
3.3. Viremic Phase of AHSV-LAV Combination-1-Vaccinated Horses
3.4. Detection of Immune Responses Against AHSV Using bELISA
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
Abbreviations
AHS | African Horse Sickness |
AHSV | African Horse Sickness Virus |
bELISA | Blocking ELISA |
BLASTn | Nucleotide BLAST |
bp | Base Pair |
BP | Blocking Percentage |
cDNA | Complementary Deoxyribonucleic Acid |
CES | Capillary Electrophoresis Sequencing |
Ct | Cycle Threshold |
DLD | Department of Livestock Development |
DNA | Deoxyribonucleic Acid |
DNase | Deoxyribonuclease |
dNTP | Deoxyribonucleotide Triphosphate |
EDTA | Ethylenediaminetetraacetic Acid |
ELISA | Enzyme-linked Immunosorbent Assay |
KVDC | Kamphaeng Saen Veterinary Diagnostic Center |
LAV | Live attenuated Vaccine |
Mab | Monoclonal Antibody |
NIAH | National Institute of Animal Health |
NS | Viral Non-structural Protein |
OBP | Onderstepoort Biological Products |
OD | Optical Density |
PCR | Polymerase Chain Reaction |
RFU | Relative Fluorescence Unit |
RNA | Ribonucleic Acid |
RNase | Ribonuclease |
Rpm | Revolutions Per Minute |
RT | Reverse Transcription |
RT-PCR | Reverse Transcription Polymerase Chain Reaction |
SNT | Serum Neutralization Test |
Tm | Melting Temperature |
U | Unit |
VP | Viral Structural Protein |
WOAH | World Organisation for Animal Health |
°C | Degree Celsius |
mM | Millimolar |
ml | Milliliter |
µL | Microliter |
μM | Micromolar |
References
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Primers | Sequences (5′ → 3′) | Purpose | Target | PCR Product Size (bp) |
---|---|---|---|---|
VP7_F | CGC-GAT-AGC-AGC-AAG-AGC-C | Group-specific amplification | VP7 | 256 |
VP7_R | GTT-GCC-AAC-GCC-TGA-TCA-TA | |||
VP5_F | GTT-TAT-TTT-TCC-AGA-AGC-CAT-GGG-TAA-ATT-C | Cloning | VP5 | 1564 |
VP5_R | GTA-AGT-GTT-TTT-CCC-GCC-CAC-AGG-CTC-C | |||
OP1_F | TCG-CAT-CTC-AAG-GTT-GC | First round of nested PCR (outer primers) | VP5 genes of AHSV-1 | 684 |
OP1_R | AAG-CGC-GTT-CAT-TAT-CGT-CC | |||
OP2_F | TAC-GTM-GAA-AAA-GCG-CT | First round of nested PCR (outer primers) | VP5 genes of AHSV-3 and -4 | 686 |
OP2_R | TGA-TGA-TGC-GGY-GCA-ATG | |||
ITH1_F | GCT-AGC-GGT-TGC-AAT-CAA-GTC-AAA-G | Second round of nested PCR (inner primers) | VP5 genes of Thai AHSV-1 | 547 |
ITH1_R | CAG-ATC-TGT-GTT-ATG-CAC-CAG-CTG-TAG-T | |||
IV1_F | ATA-TAA-TGC-ATG-GGG-GTG-CTG-TT | Second round of nested PCR (inner primers) | VP5 genes of AHSV-1 vaccine strain | 228 |
IV1_R | GGA-GAT-CAG-TAT-TAT-GAA-CCA-ACT-GTA-AA | |||
IV3_F | CCT-CCA-AAC-GGA-AGA-GGA-TTT-AAG-AAC-TTC | Second round of nested PCR (inner primers) | VP5 genes of AHSV-3 vaccine strain | 469 |
IV3_R | TTC-GTA-TTC-CTT-CTT-CAC-TAG-AGG-CAT-G | |||
IV4_F | GTT-ACA-AAC-AGA-GGA-AGA-TTT-GCG-GAC-ACG | Second round of nested PCR (inner primers) | VP5 genes of AHSV-4 vaccine strain | 437 |
IV4_R | CAT-CGA-TTA-CGT-GCT-GCG-TTT-CTA-CG |
Strain | Accession no. | Location | Year | %Similarity to AHSV2020/043 VP5 |
---|---|---|---|---|
Field AHSV-1 | MT586217 | Thailand | 2020 | 99.9 |
Vaccine AHSV-1 | KT030334 | South Africa | 1998 | 85.5 |
Vaccine AHSV-3 | KT030344 | South Africa | 1998 | 72.4 |
Vaccine AHSV-4 | KT030354 | South Africa | 1998 | 71.1 |
Field AHSV-2 | OM289934 | Kenya | 2015 | 99.5 |
Field AHSV-2 | KP009636 | South Africa | 2007 | 99.1 |
Field AHSV-2 | FJ196589 | Nigeria | 2008 | 99 |
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Pipitpornsirikul, P.; Thangthamniyom, N.; Laikul, A.; Songkasupa, T.; Pathomsakulwong, W.; Apichaimongkonkun, T.; Kasemsuwan, S.; E-kobon, T.; Lekcharoensuk, P. The Viremic Phase and Humoral Immune Response Against African Horse Sickness Virus That Emerged in Thailand in 2020. Vet. Sci. 2025, 12, 878. https://doi.org/10.3390/vetsci12090878
Pipitpornsirikul P, Thangthamniyom N, Laikul A, Songkasupa T, Pathomsakulwong W, Apichaimongkonkun T, Kasemsuwan S, E-kobon T, Lekcharoensuk P. The Viremic Phase and Humoral Immune Response Against African Horse Sickness Virus That Emerged in Thailand in 2020. Veterinary Sciences. 2025; 12(9):878. https://doi.org/10.3390/vetsci12090878
Chicago/Turabian StylePipitpornsirikul, Paphavee, Nattarat Thangthamniyom, Aree Laikul, Tapanut Songkasupa, Watcharapol Pathomsakulwong, Tawanhathai Apichaimongkonkun, Suwicha Kasemsuwan, Teerasak E-kobon, and Porntippa Lekcharoensuk. 2025. "The Viremic Phase and Humoral Immune Response Against African Horse Sickness Virus That Emerged in Thailand in 2020" Veterinary Sciences 12, no. 9: 878. https://doi.org/10.3390/vetsci12090878
APA StylePipitpornsirikul, P., Thangthamniyom, N., Laikul, A., Songkasupa, T., Pathomsakulwong, W., Apichaimongkonkun, T., Kasemsuwan, S., E-kobon, T., & Lekcharoensuk, P. (2025). The Viremic Phase and Humoral Immune Response Against African Horse Sickness Virus That Emerged in Thailand in 2020. Veterinary Sciences, 12(9), 878. https://doi.org/10.3390/vetsci12090878