Submitted to: Avian Diseases
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 2/15/2007
Publication Date: 9/1/2007
Citation: Silva, R.F., Fadly, A.M., Taylor, S.P. 2007. Development of a polymerase chain reaction to differentiate avian leukosis virus (ALV) subgroups: detection of an ALV contaminate in a commercial Marek's disease vaccine. Avian Diseases. 51(3):663-667. Interpretive Summary: Avian leukosis viruses (ALV) commonly infect chickens. ALVs have been divided into 6 subgroups (A, B, C, D, E, and J). The subgroup E viruses are present in nearly all chickens and do not usually cause disease. The other 5 subgroups can cause disease in susceptible chickens. It can be difficult to differentiate between the subgroups because the different subgroups are so closely related. We report developing a simple and fast assay for identifying and differentiating the different subgroups. We used the assay to demonstrate that two commercial vaccines for Marek’s disease were contaminated with a subgroup A ALV. We DNA sequenced a portion of the contaminating virus and by comparing the sequence to other ALVs, we confirmed that the virus was a subgroup A ALV. DNA sequencing also indicated that the contaminant virus contained a region that was unlike other subgroup A viruses but was very similar to a region in subgroup E viruses. The subgroup E-like portion of the virus made it grow very slow in cell culture and is probably the reason that the virus was not initially detected when the vaccines were screened for contaminants.
Technical Abstract: Avian leukosis viruses (ALVs) are common in many poultry flocks and can be detected by using an ELISA assay or any other test designed to identify the viral antigen p27. However, endogenous retroviruses, expressing p27, are often present and can be confused with exogenous ALVs. A more specific and informative assay is to target the variable envelope glycoprotein gene (gp85) that is the basis for dividing ALVs into their different subgroups. We designed PCR primers that would specifically detect and amplify viruses from each of the six ALV subgroups, A, B, C, D, E, and J. Subgroup B and D envelopes are related and our B-specific primers also amplified subgroup D viruses. We also designed a set of common primers to amplify any ALV subgroup virus. To demonstrate the usefulness of these primers, we obtained from the Center for Veterinary Biologics in Iowa (CVB), culture supernatant from CEFs infected with an ALV that was found to be a contaminant in two commercial Marek's disease vaccines. Using our PCR primers, we demonstrate that the contaminant was a subgroup A ALV. We cloned and sequenced a portion of the envelope gene and confirmed that the ALV was a subgroup A virus. Unlike typical subgroup A viruses, the contaminant ALV grew very slowly in cell culture. We also cloned and sequenced a portion of the LTR from the contaminant virus. The LTR was found to be similar to LTRs found in endogenous ALVs (subgroup E) and very dissimilar to LTRs normally found in subgroup A viruses. The E-like LTR probably explains why the contaminant grew so poorly in cell culture.