Establishment of a TaqMan-based real-time quantitative PCR method for detection of exogenous fowl adenovirus type Ⅰ, type Ⅲ and avian leukosis virus in human cold adapted live attenuated influenza vaccines

Min Wu; Baihui Zhi; Rui Sheng; Entong Zhou; Qian Zhou; Jiemin Li; Xiaoshu Fu; Yanchun Wu; Yunchang Zhang; Pingjie Sun; Wei Jia; Liang Shi; Dawei Liu; Hui Zhao; Weiheng Su

doi:10.1016/j.heliyon.2023.e23033

Establishment of a TaqMan-based real-time quantitative PCR method for detection of exogenous fowl adenovirus type Ⅰ, type Ⅲ and avian leukosis virus in human cold adapted live attenuated influenza vaccines

Heliyon. 2023 Nov 29;9(12):e23033. doi: 10.1016/j.heliyon.2023.e23033. eCollection 2023 Dec.

Authors

Min Wu¹, Baihui Zhi², Rui Sheng¹, Entong Zhou¹, Qian Zhou³, Jiemin Li¹, Xiaoshu Fu¹, Yanchun Wu², Yunchang Zhang², Pingjie Sun², Wei Jia², Liang Shi², Dawei Liu², Hui Zhao⁴, Weiheng Su^{1

5}

Affiliations

¹ National Engineering Laboratory for AIDS Vaccine, School of Life Sciences, Jilin University, Changchun, 130012, China.
² BCHT Biotechnology Company, Changchun, 130012, China.
³ Beijing Vital River Laboratory Animal Technology Company, Beijing, 100012, China.
⁴ National Institutes for Food and Drug Control, Beijing, 102629, China.
⁵ Key Laboratory for Molecular Enzymology and Engineering of the Ministry of Education, School of Life Sciences, Jilin University, Changchun, 130012, China.

Abstract

Cold adapted live attenuated influenza vaccines can effectively prevent human disease and death caused by influenza virus. Since chicken embryos are used as the culture substrate for the large-scale production of influenza vaccines, cold adapted live attenuated influenza vaccines may be contaminated by exogenous avian viruses. Rapid and sensitive methods such as TaqMan-based quantitative PCR are needed for the detection of exogenous avian viruses during cold adapted live attenuated influenza vaccines production. In this study, a TaqMan-based quantitative PCR method was established for the detection of three common exogenous avian viruses, including fowl adenovirus type I, type Ⅲ and avian leukosis virus. Avian virus-encoding plasmids purified in high-performance liquid chromatography were essential for sensitivity analysis. The sensitivity reached 1 copy per reaction for each of the avian virus plasmids. Standard curves showed a strong linear relationship. The TaqMan-based quantitative PCR method had high specificity and no cross-reactivity with other irrelevant viruses. Furthermore, the established TaqMan-based quantitative PCR can effectively detect 0.1 TCID₅₀ of each avian virus without or with interference from the influenza virus nucleic acid. Ultimately, this method was used to test three master seed lots of monovalent cold adapted live attenuated influenza vaccine, and the results showed that no fowl adenovirus type I, type Ⅲ or avian leukosis virus contamination, which were consistent with serological methods. The TaqMan-based quantitative PCR method for the determination of extraneous avian viruses in cold adapted live attenuated influenza vaccines met the requirement for both conventional and emergency inspection on cold adapted live attenuated influenza vaccines.

Keywords: Avian leukosis virus (ALV); Cold adapted live attenuated influenza vaccines (CA-LAIVs); Exogenous avian virus; Fowl adenovirus type Ⅰ (FAdV Ⅰ); Fowl adenovirus type Ⅲ (FAdV Ⅲ); TaqMan-based quantitative PCR (qPCR).