Real-time PCR-based infectivity assay for the titration of turkey hemorrhagic enteritis virus, an adenovirus, in live vaccines.
The current in vitro titration method for turkey hemorrhagic enteritis virus (THEV) is the end-point dilution assay (EPD) in suspension cell culture (CC). This assay is subjective and results in high variability among vaccine lots. In this study, a new in vitro infectivity method combining a SYBR Green I-based qPCR assay and CC was developed for titration of live hemorrhagic enteritis (HE) CC vaccines. The qPCR was used to determine the virus genome copy number (vGCN) of the internalized virus particles following inoculation of susceptible RP19 cells with 1 vaccine label dose. The measured vGCN represents the number of infectious viral particles (IVP) per 1 dose. This method was used to compare 9 vaccine lots from 3 companies in the United States. Significant lot-to-lot variations within the same company and among the various companies were found in genomic and qPCR-based infectious titer per label dose. A positive linear relationship was found between qPCR infectious titer and genomic titer. Further, considerable variations in CCID50titers were found among tested vaccine lots, indicating the high variability of the current titration methods. The new method provides an alternative to classical titration assays and can help reduce variation among HE vaccine products.
Department of Population Health Sciences, Virginia-Maryland College of Veterinary Medicine, Virginia Tech, 205 Duck Pond Drive, Blacksburg, VA 24061-0442, USA,; Poultry Production Department, Faculty of Agriculture, Alexandria University, Alexandria, Egypt; Center for Molecular Medicine and Infectious Diseases, Virginia-Maryland College of Veterinary Medicine, Virginia Tech, 1410 Prices Fork Road, Blacksburg, VA 24061-0342, USA,. Electronic address: hmahsoub@vt.edu.
Hassan M
HM
Mahsoub
Department of Population Health Sciences, Virginia-Maryland College of Veterinary Medicine, Virginia Tech, 205 Duck Pond Drive, Blacksburg, VA 24061-0442, USA,; Center for Molecular Medicine and Infectious Diseases, Virginia-Maryland College of Veterinary Medicine, Virginia Tech, 1410 Prices Fork Road, Blacksburg, VA 24061-0342, USA,. Electronic address: nievans@vt.edu.
Nicholas P
NP
Evans
Center for Molecular Medicine and Infectious Diseases, Virginia-Maryland College of Veterinary Medicine, Virginia Tech, 1410 Prices Fork Road, Blacksburg, VA 24061-0342, USA,. Electronic address: beachnm@gmail.com.
Nathan M
NM
Beach
Department of Pathology and Pathobiology, Virginia-Maryland College of Veterinary Medicine, Virginia Tech, 205 Duck Pond Drive, Blacksburg, VA 24061-0442, USA. Electronic address: lyuan@vt.edu.
Lijuan
L
Yuan
Department of Pathology and Pathobiology, Virginia-Maryland College of Veterinary Medicine, Virginia Tech, 205 Duck Pond Drive, Blacksburg, VA 24061-0442, USA. Electronic address: kzimmerm@vt.edu.
Kurt
K
Zimmerman
Department of Population Health Sciences, Virginia-Maryland College of Veterinary Medicine, Virginia Tech, 205 Duck Pond Drive, Blacksburg, VA 24061-0442, USA,; Center for Molecular Medicine and Infectious Diseases, Virginia-Maryland College of Veterinary Medicine, Virginia Tech, 1410 Prices Fork Road, Blacksburg, VA 24061-0342, USA,. Electronic address: pierson@vt.edu.
Frank W
FW
Pierson
D000256:Adenoviridae / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification*; D000818:Animals; D000956:Antigens, Viral / Q000032:analysis* / Q000235:genetics; D018929:Cell Culture Techniques; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D014422:Turkeys / Q000821:virology; D014613:Vaccines, Attenuated; D014765:Viral Vaccines
D000956:Antigens, Viral; D014613:Vaccines, Attenuated; D014765:Viral Vaccines
Cell culture; Hemorrhagic enteritis; PCR; Turkey; Vaccine titration