Journal Article

Establishment of a real-time fluorescent quantitative PCR detection method and phylogenetic analysis of BoAHV-1

Li, Dongli, Cui, Zhenzhen, Liu, Yingyu, Xu, Lihui, Liu, Fei, Li, Jianming, Leng, Xue, Diao, Naichao, Ge, Guiyang, Gong, Qinglong, Du, Rui, Shi, Kun

2024

Overview

Background Infectious bovine rhinotracheitis  (IBR), caused by Bovine alphaherpesvirus-1 (BoAHV-1), is an acute, highly contagious disease primarily characterized by respiratory tract lesions in infected cattle. Due to its severe pathological damage and extensive transmission, it results in significant economic losses in the cattle industry. Accurate detection of BoAHV-1 is of paramount importance. In this study, we developed a real-time fluorescent quantitative PCR detection method for detecting BoAHV-1 infections. Utilizing this method, we tested clinical samples and successfully identified and isolated a strain of BoAHV-1.1 from positive samples. Subsequently, we conducted a genetic evolution analysis on the isolate strain’s gC, TK, gG, gD, and gE genes. Results The study developed a real-time quantitative PCR detection method using SYBR Green II, achieving a detection limit of 7.8 × 10 1 DNA copies/μL. Specificity and repeatability analyses demonstrated no cross-reactivity with other related pathogens, highlighting excellent repeatability. Using this method, 15 out of 86 clinical nasal swab samples from cattle were found to be positive (17.44%), which was higher than the results obtained from conventional PCR detection (13.95%, 12/86). The homology analysis and phylogenetic tree analysis of the gC, TK, gG, gD, and gE genes of the isolated strain indicate that the JL5 strain shares high homology with the BoAHV-1.1 reference strains. Amino acid sequence analysis revealed that gC, gE, and gG each had two amino acid mutations, while the TK gene had one synonymous mutation and one H to Y mutation, with no amino acid mutations observed in the gD gene. Phylogenetic tree analysis indicated that the JL5 strain belongs to the BoAHV-1.1 genotype and is closely related to American strains such as C33, C14, and C28. Conclusions The established real-time fluorescent quantitative PCR detection method exhibits good repeatability, specificity, and sensitivity. Furthermore, genetic evolution analysis of the isolated BoAHV-1 JL-5 strain indicates that it belongs to the BoAHV-1.1 subtype. These findings provide a foundation and data for the detection, prevention, and control Infectious Bovine Rhinotracheitis.

• Publisher: BioMed Central,BioMed Central Ltd,Springer Nature B.V,BMC
• Subject: Alphaherpesvirinae - classification / Alphaherpesvirinae - genetics / Alphaherpesvirinae - isolation & purification / Amino acid sequence / amino acid sequences / Amino acids / Animals / Bacterial infections / BoAHV-1 / Bovine rhinotracheitis / Cattle / Cattle industry / cross reaction / Cross-reactivity / detection limit / Disease prevention / Diseases / DNA / Evolution & development / fluorescence / Genes / Genetic analysis / Genetic aspects / genotype / Genotypes / Herpesviruses / Homology / Infections / infectious bovine rhinotracheitis / Infectious Bovine Rhinotracheitis - virology / Medicine / Medicine & Public Health / Methods / Mutation / nose / Pathogens / Phylogenetics / Phylogenic analysis / Phylogeny / Polymerase chain reaction / quantitative polymerase chain reaction / Real-Time Polymerase Chain Reaction - methods / Real-Time Polymerase Chain Reaction - standards / Reproducibility / Respiratory diseases / RT‒qPCR / Sensitivity and Specificity / Sequence analysis / Specimen Handling - veterinary / Strains (organisms) / Testing / TK gene / Transgenics / Vaccines / Veterinary Medicine/Veterinary Science / Viruses / Zoology