Real-time PCR-based infectivity assay for the titration of turkey hemorrhagic enteritis virus, an adenovirus, in live vaccines

Highlights

• A new infectivity titration method combining qPCR with cell culture was developed for turkey hemorrhagic enteritis virus.

• The method was used for titration of 9 lots of hemorrhagic enteritis vaccine.

• Significant variations in genomic and infectious titers were found.

• Genomic titer of HE vaccines can serve as an indicator of qPCR infectious titer.

• The method is a useful alternative for current traditional methods.

Abstract

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 CCID₅₀ titers 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.

Introduction

Turkey hemorrhagic enteritis virus (THEV) is the etiologic agent of the hemorrhagic enteritis (HE) disease in turkeys. This virus, also known as turkey adenovirus 3, belongs to the family Adenoviridae, genus Siadenovirus, species Turkey adenovirus A (Davison and Harrach, 2011). Disease caused by virulent strains of THEV can be prevented by vaccination of 4½-6 weeks-of-age turkey poults with naturally occurring avirulent THEV strains (Domermuth et al., 1977, Larsen et al., 1985, Newberry et al., 1993). USDA-licensed live vaccine products propagated in cell culture (CC) are available to prevent HE. Vaccination of poults with 100 CCID per bird of CC-propagated HE vaccine is sufficient for protection against disease (Fadly and Nazerian, 1984).

Splenomegaly and a transient period of immunosuppression is caused by both virulent and avirulent (vaccine) strains of THEV in susceptible birds (Pierson and Fitzgerald, 2008, van den Hurk et al., 1994). The virus targets the spleen where it mainly replicates in B lymphocytes and induces their apoptosis. This is followed by a reduction in the numbers of IgM-bearing B cells in the spleen and peripheral blood at days 2, 3, 4, and 9 after infection, which temporarily suppresses the birds immune system and makes them vulnerable to potential secondary infections (e.g., E. coli) that can lead to high mortality (up to 60%) (Pierson et al., 1996, Rautenschlein et al., 2000, Saunders et al., 1993, Suresh and Sharma, 1995, Suresh and Sharma, 1996). Poults inoculated with increasing doses of HE vaccine showed a dose-dependent increase in their relative spleen weight (Fadly and Nazerian, 1984). This indicates that the level of immunosuppression in vaccinated birds may also be dose-dependent. Therefore, for efficacious use of HE vaccines without a deleterious immune response, methods for determining an accurate titer per label dose are necessary.

Currently, two EPD assays are available for use to determine THEV infectivity titers in live vaccines. The first is an in vivo titration in 5–6 week old turkeys that are THEV-seronegative (Domermuth et al., 1977). This method is labor-intensive and relatively expensive to perform as a routine titration method. Birds are inoculated with serial dilutions of infectious material and assessed for splenic enlargement/mottling 3–5 days later depending on the route of inoculation. This method is used to calculate the “poult infectious dose”. The second method is an in vitro endpoint dilution in MDTC-RP19, B-lymphoblastoid cells (RP19 cells) (Nazerian et al., 1982, Sharma, 1994). This method relies solely on visual evaluation of the cytopathic effect (CPE) observed for infected cells (i.e., enlargement and ballooning of cells), the only overt manifestation of infection and viral replication. Because RP19 cells grow in suspension and determining CPE is subjective, the sensitivity and efficiency of this method is lower than those employing monolayer cultures (Nazerian and Fadly, 1982, Smither et al., 2013). Modified protocols that incorporate immunostaining to confirm the CPE scores in infected RP19 cells were developed, but they add to the cost and time necessary to complete the assay (Nazerian and Fadly, 1987, Rautenschlein and Sharma, 1999, van den Hurk, 1990). Other standard in vitro titration methods (e.g., plaque formation and focus formation assays) cannot be applied to THEV because there are no susceptible adherent cell lines (Nazerian and Fadly, 1987). As with other traditional titration methods, these assays are laborious, require a long post-inoculation incubation period, suffer from low-throughput, poor reproducibility and sensitivity (Forsey et al., 1992, LaBarre and Lowy, 2001, Mena et al., 2003). The inherent inaccuracy associated with these traditional titration methods potentially results in poor field performance of vaccines.

Since current methods are not available to accurately titrate THEV, there is a need for a quantitative virus titration method. In recent years a new trend has emerged for quantification of infectious virus particles by infectivity methods that integrate qPCR technology, both SYBR Green and TaqMan, with cell culture systems. The use of such methods throughout the multiple phases of vaccine development and assessment has been recently reviewed (Wolf et al., 2007), highlighting their importance relative to current vaccine production technologies. qPCR-based potency estimation and infectivity titration have been successfully employed for a variety of human and animal DNA and RNA viruses including orf virus (genus: Parapoxvirus, family: Poxviridae), adeno-associated viruses (Dependoparvovirus, Parvoviridae), camelpox and buffalopox viruses (Orthopoxvirus, Poxviridae), rotaviruses (Rotavirus, Reoviridae), measles virus (Morbillivirus, Paramyxoviridae), mumps virus (Rubulavirus, Paramyxoviridae), rubella virus (Rubivirus, Togaviridae), and adenoviruses (Mastadenovirus, Adenoviridae) (Ammour et al., 2013, Bora et al., 2012, Mayginnes et al., 2006, Prabhu et al., 2012, Ranheim et al., 2006, Rohr et al., 2005, Wang et al., 2005). The aim of the present study was to establish a rapid and quantitative method for the infectivity titration of THEV in live vaccines using a combination of RP19 cell culture and qPCR assay.