Development and Application of Real-Time PCR for Detection of Subgroup J Avian Leukosis Virus

Abstract

Subgroup J avian leukosis virus (ALV-J) is an avian retrovirus that causes severe economic losses in the poultry industry. The early identification and removal of virus-shedding birds are important to reduce the spread of congenital and contact infections. In this study, a TaqMan-based real-time PCR method for the rapid detection and quantification of ALV-J with proviral DNA was developed. This method exhibited a high specificity for ALV-J. Moreover, the detection limit was as low as 10 viral DNA copies. The coefficients of variation (CVs) of both interassay and intra-assay reproducibility were less than 1%. The growth curves of ALV-J in DF-1 cells were measured by real-time PCR, yielding a trend line similar to those determined by 50% tissue culture infective dose (TCID 50 ) and p27 antigen detection. Tissue samples suspected of ALV infection were evaluated using real-time PCR, virus isolation, and routine PCR, and the positivity rates were 60.1%, 41.6% and 44.5%, respectively. Our data indicated that the real-time PCR method provides a sensitive, specific, and reproducible diagnostic tool for the identification and quantification of ALV-J for clinical diagnosis and in laboratory research. Avian leukosis viruses (ALVs) are members of the Alpharetrovirus genus of the family Retroviridae that are associated with a variety of neoplasms, including lymphoid and myeloid leukoses. ALVs are prevalent in poultry flocks worldwide and cause serious economic losses due to decreased egg production and quality, tumor mortality, and low growth rate. ALV isolates from chickens are classified into subgroups A, B, C, D, and E and the recently identified subgroup J according to host range, viral envelope glycoprotein properties, and cross-neutralization patterns (1). Subgroup A and B ALVs occur as common pathogenic exogenous viruses, and subgroup C and D ALVs have been rarely reported in the field. Subgroup E viruses are endogenous leukosis viruses with low pathogenicity. Subgroup J ALV (ALV-J) is an exogenous virus that causes myeloid leukosis (ML) in chickens. ALV-J was first isolated from meat-type chickens and was designated subgroup J due to its distinct envelope characteristics (2). The env gene sequence of the ALV-J prototype (HPRS-103 strain) is considerably different from those of other subgroups. However, it shares high identities (75 to 97%) with env-like sequences from the endogenous avian retrovirus (EAV) family members, suggesting that it evolved by recombination with the env-like sequences of this family (3, 4).ALV-J was first isolated from broiler chickens with myeloid leukosis in the United Kingdom. Since its first report, ALV-J infection has been widely detected in multiple countries in the Americas, Asia, and Oceania (5-7). Although the virus has a broad host range and all chicken lines tested have been susceptible to infection (2, 8), no field cases of ALV-J infection and tumors in layer chickens were reported until 2004 (9). In recent years, cases of ALV-J infection and tumors have been widely reported for co...

Development and Application of a Multiplex PCR Method for Rapid Differential Detection of Subgroup A, B, and J Avian Leukosis Viruses

“…Real-time PCR and immunofluorescence assays have been developed for antigen detection and the differentiation of endogenous and exogenous ALVs. Nevertheless, both of these techniques require sophisticated instrumentation (such as quantitative fluorescence PCR machines and fluorescence microscopes) and cannot be used widely in the field (15,16). Virus isolation in cell culture is often used as the gold standard.…”

“…An aliquot of the supernatant of all samples was used to extract proviral DNA, which was utilized as a template for mPCR and routine PCR detection. The remaining supernatant was passed through 0.22-m filters to carry out virus isolation (16).…”

Rapid detection of the common avian leukosis virus subgroups by real-time loop-mediated isothermal amplification

“…It is too difficult to apply for the numerous smaller or mid-sized poultry breeding companies in China as opposed to the fewer, larger firms in the USA and many European countries. The p27 antigen ELISA assay is routinely used for the ALV diagnosis, but it also is expensive and timeconsuming [28]. These are the reasons we have developed a specific, sensitive, inexpensive and real-time monitored LAMP assay for the detection of major ALV subgroups infected in chickens.…”

Avian leukosis virus subgroup J infection influencing composition of gut microbiota within chicken

“…Water was supplied via two 'on-demand' nipples per cage. Followed by a method [34], chickens were divided into group of viral control (A group) or naturally ALV-J infected (B group) with 12 individuals in each group, respectively. All chickens were euthanized by cervical dislocation.…”