The reverse transcriptase activity in cell-free medium of chicken embryo fibroblast cultures is not associated with a replication-competent retrovirus

305

218

Metagenomics and a panmicrobial microarray were used to examine eight live-attenuated viral vaccines. Viral nucleic acids in trivalent oral poliovirus (OPV), rubella, measles, yellow fever, varicella-zoster, multivalent measles/mumps/rubella, and two rotavirus live vaccines were partially purified, randomly amplified, and pyrosequenced. Over half a million sequence reads were generated covering from 20 to 99% of the attenuated viral genomes at depths reaching up to 8,000 reads per nucleotides. Mutations and minority variants, relative to vaccine strains, not known to affect attenuation were detected in OPV, mumps virus, and varicella-zoster virus. The anticipated detection of endogenous retroviral sequences from the producer avian and primate cells was confirmed. Avian leukosis virus (ALV), previously shown to be noninfectious for humans, was present as RNA in viral particles, while simian retrovirus (SRV) was present as genetically defective DNA. Rotarix, an orally administered rotavirus vaccine, contained porcine circovirus-1 (PCV1), a highly prevalent nonpathogenic pig virus, which has not been shown to be infectious in humans. Hybridization of vaccine nucleic acids to a panmicrobial microarray confirmed the presence of endogenous retroviral and PCV1 nucleic acids. Deep sequencing and microarrays can therefore detect attenuated virus sequence changes, minority variants, and adventitious viruses and help maintain the current safety record of live-attenuated viral vaccines.

Section: Vol 84 2010 Adventitious Virus In Live-attenuated Vaccine mentioning

confidence: 99%

Viral Nucleic Acids in Live-Attenuated Vaccines: Detection of Minority Variants and an Adventitious Virus

Victoria

Wang²,

Jones

et al. 2010

305

218

“…The majority of these sequences are defective; however, some may produce infectious retroviruses (9,11,16,71,78). In general, newly acquired endogenous retroviral sequences are more likely to be associated with an infectious virus, whereas ancient sequences may be transcriptionally active but defective for virus production (86) or produce noninfectious particles (38). In rodents, endogenous retroviruses can become activated in animals, as a consequence of age (4), or in cell lines, either spontaneously by long-term culture passage (2,49,63) or by treatment with a variety of inducers, including biological, immunological, and chemical agents (1,13,16,25,39,47,50).…”

mentioning

confidence: 99%

Chemical Induction of Endogenous Retrovirus Particles from the Vero Cell Line of African Green Monkeys

et al. 2011

“…While reassuring, these data are limited by the absence of information regarding whether these vaccine recipients were exposed to infectious ALV-E and whether humans are indeed susceptible to ALV-E infections. Two recent studies showed that inoculation of different human cell types with CEF supernatants did not result in ALV infection or in detectable RT activity (29,37). However, it is not known whether the CEF supernatants used contained replication-competent ALV-E.…”

Section: Discussionmentioning

confidence: 99%

Characterization of Endogenous Avian Leukosis Viruses in Chicken Embryonic Fibroblast Substrates Used in Production of Measles and Mumps Vaccines

Johnson

Heneine

2001

Previous findings of low levels of reverse transcriptase (RT) activity in chick cell-derived measles and mumps vaccines showed this activity to be associated with virus particles containing RNA of both subgroup E endogenous avian leukosis viruses (ALV-E) and endogenous avian viruses (EAV). These particles originate from chicken embryonic fibroblast (CEF) substrates used for propagating vaccine strains. To better characterize vaccine-associated ALV-E, we examined the endogenous ALV proviruses (ev loci) present in a White Leghorn CEF substrate pool by restriction fragment length polymorphism. Five ev loci were detected, ev-1, ev-3, ev-6, ev-18, andev-19. Both ev-18 and ev-19 can express infectious ALV-E, while ev-1, ev-3, and ev-6 are defective. We analyzed the full-length sequence of ev-1 and identified an adenosine insertion within the pol RT-␤ region at position 5026, which results in a truncated RT-␤ and integrase. We defined the 1,692-bp deletion in the gag-pol region of ev-3, and we found that in ev-6, sequences from the 5 long terminal repeat to the 5 pol region were absent. Based on the sequences of the ev loci, RT-PCR assays were developed to examine expression of ALV-E particles (EV) in CEF supernatants. Both ev-1-and ev-3-like RNA sequences were identified, as well as two other RNA sequences with intact pol regions, presumably of ev-18 and ev-19 origin. Inoculation of susceptible quail fibroblasts with CEF culture supernatants from both 5-azacytidine-induced and noninduced CEF led to ALV infection, confirming the presence of infectious ALV-E. Our data demonstrate that both defective and nondefective ev loci can be present in CEF vaccine substrates and suggest that both ev classes may contribute to the ALV present in vaccines.Reverse transcriptase (RT) activity, an indication of the presence of retroviruses, was recently detected in chick cellderived live, attenuated vaccines including those produced by European and U.S. manufacturers for measles, mumps, and yellow fever (8,32,41). Chicken embryos and chicken embryonic fibroblasts (CEFs) from controlled breeding flocks are used in vaccine manufacture to propagate high-titer attenuated vaccine inocula. The use of chicken embryos and CEF in vaccine manufacturing requires that they be derived from closed, specific-pathogen-free source chickens that are free of known exogenous retroviral chicken pathogens, including the reticuloendotheliosis virus and the avian leukosis virus (ALV) groups (45).Investigations of the origin of RT activity in the measles vaccine found evidence of particles containing endogenous avian virus (EAV) RNA in a vaccine manufactured in Europe (44), while evidence of both EAV and endogenous avian leukosis virus (ALV-E) was found in a vaccine made in the United States (41). While vaccine manufacturing regulations require elimination of exogenous retroviral infections from source chickens, these regulations do not address the presence of endogenous retroviruses because such particles were not previously known to be associated with chick c...