Endogenous Avian Leukosis Virus
Endogenous Avian Leukosis Virus
The identification of endogenous avian leukosis virus (ALV) and endogenous avian retrovirus (EAV) in chick cell-derived measles and mumps vaccines in current use has raised concern about transmission of these retroviruses to vaccine recipients. We used serologic and molecular methods to analyze specimens from 206 recipients of measles, mumps, and rubella vaccine for evidence of infection with ALV and EAV. A Western blot assay for detecting antibodies to endogenous ALV was developed and validated. All serum samples were negative for antibodies to endogenous ALV by Western blot analysis. Peripheral blood lymphocyte samples from 100 vaccinees were further tested by polymerase chain reaction for both ALV and EAV proviral sequences; all were negative. Matching serum samples were tested by reverse transcriptase polymerase chain reaction for ALV and EAV RNA, and all 100 samples were negative, providing no evidence of viremia. These findings do not indicate the presence of either ALV or EAV infection in MMR vaccine recipients and provide support for current immunization policies.
Vaccines effectively reduce and prevent death and disease from many viral infections. However, vaccine production occasionally has been complicated by inadvertent contamination with adventitious agents that may have originated from cell substrates used to propagate vaccine strains. Examples of such contamination include simian virus in early polio vaccines grown on monkey kidney cells and avian leukosis virus (ALV) in yellow fever vaccines propagated in chick embryos. Hepatitis B virus has also been identified in yellow fever vaccines produced by using pooled human serum as a stabilizing agent. Exposure of vaccine recipients to contaminated vaccines has been associated with effects ranging from benign to demonstrable transmission of infection, with or without subsequent disease.
Reverse transcriptase (RT) activity, an indicator of retroviruses, has recently been detected by sensitive polymerase chain reaction (PCR)-based RT assays in currently used vaccines produced in chick embryo fibroblasts or embryonated eggs. The RT-positive vaccines include measles, mumps, and yellow fever vaccines produced by several manufacturers in Europe and the United States. RT activity was detected in the vaccines despite strict manufacturing practices requiring that chick embryos and embryo fibroblasts be derived from closed, specific-pathogen-free chicken flocks. Such chickens are screened for known pathogens, including two exogenous avian retroviruses: reticuloendotheliosis virus and ALV.
The origin of RT activity in measles vaccines was examined in two recent studies. RT activity in a vaccine manufactured in Europe was associated with particles containing endogenous avian virus (EAV) RNA. In the second study, we examined measles vaccines from a U.S. manufacturer and found evidence of both EAV and endogenous ALV: we detected particle-associated ALV and EAV-0 RNA sequences in both vaccine and chick embryo fibroblast supernatants and demonstrated neutralization of RT activity in vaccines by anti-ALV RT antibodies. In addition, we observed ALV-like particles by electron microscopy in culture supernatants from chick embryo fibroblasts that had not been inoculated with vaccine viruses.
At least six subgroups of ALV (A-E and J) have been identified in chickens on the basis of differences in envelope sequences. Only subgroup E viruses are expressed from endogenous sequences that are part of the chicken germ line; all the other subgroups are exogenous. The endogenous ALV sequences are usually referred to as endogenous viral (ev) loci. At least 30 ev loci have been characterized in various chicken strains. Although endogenous ALVs are not known to be pathogenic for chickens, related species of fowl are susceptible to infection with endogenous ALV. Disease associations in these cross-species infections have not been fully investigated. Exogenous-type ALVs have been shown to cause several neoplastic diseases in infected chickens.
Less is known about EAV, which has elements distinct from but closely related to those of the ALV family of endogenous retroviruses. EAV are also present in line-0 chickens (ev-negative), which have been bred to have no ev proviruses. EAV elements exist in at least 50 copies per chicken genome.
The observed association of the RT activity of these vaccines with endogenous retroviral particles rather than exogenous retroviruses is consistent with vaccine manufacturing regulations that require exogenous ALV and reticuloendotheliosis virus infections to be eliminated from source chickens. Endogenous retroviral particles are not addressed by current manufacturing guidelines because these particles had not been associated with chick cell-derived vaccines.
The finding of RT activity in all measles vaccine lots from different manufacturers tested suggests that this occurrence is not sporadic and that vaccine recipients may be universally exposed to these retroviral particles. Surveillance for infection with ALV/EAV in vaccine recipients is important for evaluating the safety of these vaccines. This surveillance, which was recommended by the World Health Organization in a consultation meeting on RT activity in chicken cell-derived vaccines, is needed for policy decisions regarding the global use of these vaccines. We recently reported negative PCR results for ALV and EAV sequences in peripheral blood lymphocytes from 33 measles, mumps, and rubella (MMR) vaccine recipients. However, these preliminary results do not fully reflect risks for transmission of ALV and EAV because of the small number of samples analyzed and the lack of testing for antibodies and plasma viremia. We have expanded our surveillance for ALV and EAV infection in recipients of MMR vaccines and here report evidence that does not support infection with either ALV or EAV.
The identification of endogenous avian leukosis virus (ALV) and endogenous avian retrovirus (EAV) in chick cell-derived measles and mumps vaccines in current use has raised concern about transmission of these retroviruses to vaccine recipients. We used serologic and molecular methods to analyze specimens from 206 recipients of measles, mumps, and rubella vaccine for evidence of infection with ALV and EAV. A Western blot assay for detecting antibodies to endogenous ALV was developed and validated. All serum samples were negative for antibodies to endogenous ALV by Western blot analysis. Peripheral blood lymphocyte samples from 100 vaccinees were further tested by polymerase chain reaction for both ALV and EAV proviral sequences; all were negative. Matching serum samples were tested by reverse transcriptase polymerase chain reaction for ALV and EAV RNA, and all 100 samples were negative, providing no evidence of viremia. These findings do not indicate the presence of either ALV or EAV infection in MMR vaccine recipients and provide support for current immunization policies.
Vaccines effectively reduce and prevent death and disease from many viral infections. However, vaccine production occasionally has been complicated by inadvertent contamination with adventitious agents that may have originated from cell substrates used to propagate vaccine strains. Examples of such contamination include simian virus in early polio vaccines grown on monkey kidney cells and avian leukosis virus (ALV) in yellow fever vaccines propagated in chick embryos. Hepatitis B virus has also been identified in yellow fever vaccines produced by using pooled human serum as a stabilizing agent. Exposure of vaccine recipients to contaminated vaccines has been associated with effects ranging from benign to demonstrable transmission of infection, with or without subsequent disease.
Reverse transcriptase (RT) activity, an indicator of retroviruses, has recently been detected by sensitive polymerase chain reaction (PCR)-based RT assays in currently used vaccines produced in chick embryo fibroblasts or embryonated eggs. The RT-positive vaccines include measles, mumps, and yellow fever vaccines produced by several manufacturers in Europe and the United States. RT activity was detected in the vaccines despite strict manufacturing practices requiring that chick embryos and embryo fibroblasts be derived from closed, specific-pathogen-free chicken flocks. Such chickens are screened for known pathogens, including two exogenous avian retroviruses: reticuloendotheliosis virus and ALV.
The origin of RT activity in measles vaccines was examined in two recent studies. RT activity in a vaccine manufactured in Europe was associated with particles containing endogenous avian virus (EAV) RNA. In the second study, we examined measles vaccines from a U.S. manufacturer and found evidence of both EAV and endogenous ALV: we detected particle-associated ALV and EAV-0 RNA sequences in both vaccine and chick embryo fibroblast supernatants and demonstrated neutralization of RT activity in vaccines by anti-ALV RT antibodies. In addition, we observed ALV-like particles by electron microscopy in culture supernatants from chick embryo fibroblasts that had not been inoculated with vaccine viruses.
At least six subgroups of ALV (A-E and J) have been identified in chickens on the basis of differences in envelope sequences. Only subgroup E viruses are expressed from endogenous sequences that are part of the chicken germ line; all the other subgroups are exogenous. The endogenous ALV sequences are usually referred to as endogenous viral (ev) loci. At least 30 ev loci have been characterized in various chicken strains. Although endogenous ALVs are not known to be pathogenic for chickens, related species of fowl are susceptible to infection with endogenous ALV. Disease associations in these cross-species infections have not been fully investigated. Exogenous-type ALVs have been shown to cause several neoplastic diseases in infected chickens.
Less is known about EAV, which has elements distinct from but closely related to those of the ALV family of endogenous retroviruses. EAV are also present in line-0 chickens (ev-negative), which have been bred to have no ev proviruses. EAV elements exist in at least 50 copies per chicken genome.
The observed association of the RT activity of these vaccines with endogenous retroviral particles rather than exogenous retroviruses is consistent with vaccine manufacturing regulations that require exogenous ALV and reticuloendotheliosis virus infections to be eliminated from source chickens. Endogenous retroviral particles are not addressed by current manufacturing guidelines because these particles had not been associated with chick cell-derived vaccines.
The finding of RT activity in all measles vaccine lots from different manufacturers tested suggests that this occurrence is not sporadic and that vaccine recipients may be universally exposed to these retroviral particles. Surveillance for infection with ALV/EAV in vaccine recipients is important for evaluating the safety of these vaccines. This surveillance, which was recommended by the World Health Organization in a consultation meeting on RT activity in chicken cell-derived vaccines, is needed for policy decisions regarding the global use of these vaccines. We recently reported negative PCR results for ALV and EAV sequences in peripheral blood lymphocytes from 33 measles, mumps, and rubella (MMR) vaccine recipients. However, these preliminary results do not fully reflect risks for transmission of ALV and EAV because of the small number of samples analyzed and the lack of testing for antibodies and plasma viremia. We have expanded our surveillance for ALV and EAV infection in recipients of MMR vaccines and here report evidence that does not support infection with either ALV or EAV.
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