Growth characteristics of fowl adenovirus type 8 in a chicken hepatoma cell line

Alexander, Hazel; Huber, P; Cao, Jingxin; Krell, Peter J.; Nagy, Éva

doi:10.1016/s0166-0934(98)00062-7

Cited by 50 publications

(32 citation statements)

References 16 publications

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“…FAdV-9 (strain A-2A) and FAdV-9⌬4 were propagated and titrated in chicken hepatoma cells (CH-SAH) as described previously (22). The cells were maintained in Dulbecco's modified Eagle's medium and nutrient mixture Ham's F-12 medium (DMEM-F12) supplemented with 10% non-heat-inactivated fetal bovine serum (FBS), 2 mM L-glutamine, 100 U/ml penicillin, and 100 g/ml streptomycin.…”

Section: Methodsmentioning

confidence: 99%

Oral Inoculation of Chickens with a Candidate Fowl Adenovirus 9 Vector

Deng

Sharif

Nagy

2013

Clin Vaccine Immunol

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Fowl adenoviruses (FAdVs) are a potential alternative to human adenovirus-based vaccine vectors. Our previous studies demonstrated that a 2.4-kb region at the left end of the FAdV-9 genome is nonessential for virus replication and is suitable for the insertion or replacement of transgenes. Our in vivo study showed that the virus FAdV-9⌬4, lacking six open reading frames (ORFs) at the left end of its genome, replicates less efficiently than wild-type FAdV-9 (wtFAdV-9) in chickens that were infected intramuscularly. However, the fecal-oral route is the natural route of FAdV infection, and the oral administration of a vaccine confers some advantages compared to administration through other routes, especially when developing an adenovirus as a vaccine vector. Therefore, we sought to investigate the effects of FAdV-9 in orally inoculated chickens. In the present study, we orally inoculated specific-pathogen-free (SPF) chickens with FAdV-9 and FAdV-9⌬4 and assessed virus shedding, antibody response, and viral genome copy number and cytokine gene expression in tissues. Our data showed that FAdV-9⌬4 replicated less efficiently than did wtFAdV-9, as evidenced by reduced virus shedding in feces, lower viral genome copy number in tissues, and lower antibody response, which are consistent with the results of the intramuscular route of immunization. Furthermore, we found that both wtFAdV-9 and FAdV-9⌬4 upregulated the mRNA expression of alpha interferon (IFN-␣), IFN-␥, and interleukin-12 (IL-12). In addition, there was a trend toward downregulation of IL-10 gene expression caused by both viruses. These findings indicate that one or more of the six deleted ORFs contribute to modulating the host response against virus infection as well as virus replication in vivo.

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Section: Methodsmentioning

confidence: 99%

Oral Inoculation of Chickens with a Candidate Fowl Adenovirus 9 Vector

Deng

Sharif

Nagy

2013

Clin Vaccine Immunol

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“…FAdV-9 strain A-2A, a non-pathogenic strain, was propagated in chicken hepatoma cells (CH-SAH cell line). Virustitration and one-step growth-curve assays were also carried out in these cells as described previously (Alexander et al, 1998). In addition, the infectivity of FAdV-9D mutant viruses was determined in a quail fibroblast (QT-35) cell line and primary chicken embryo liver (CELi) cells.…”

Section: Methodsmentioning

confidence: 99%

“…Livers from 15-day-old chicken embryos were collected aseptically and processed as described by Schat & Purchase (1989) to obtain the CELi cells. CH-SAH and QT-35 cells were maintained in Dulbecco's modified Eagle's medium/nutrient mixture F-12 Ham with 10 % non-heat-inactivated fetal bovine serum as described previously (Alexander et al, 1998).…”

Section: Methodsmentioning

confidence: 99%

“…The supernatants were stored at 270 uC. Viruses were titrated as described previously (Alexander et al, 1998) and plaques were picked for virus propagation followed by DNA extraction as reported previously (Ojkic & Nagy, 2000. To confirm the deletions in the recovered viruses from cloacal swabs, virus DNAs were digested with NotI and used as templates for PCR with primers FAD-1 and FAD-2 (Supplementary Table S1), followed by sequencing.…”

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

A region at the left end of the fowl adenovirus 9 genome that is non-essential in vitro has consequences in vivo

Corredor

Nagy

2009

Journal of General Virology

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The regions at the left and right ends of fowl adenovirus (FAdV) genomes are not wellcharacterized in comparison to those of human adenoviruses. Using a series of deletion mutants, we analysed a 2.4 kb region near the left end of the FAdV-9 genome (nt 400-2782) that contains packaging-signal motifs VI and VII and open reading frames (ORFs) 0, 1, 1A, 1B, 1C and 2. Viable viruses with specific deletions in this region had wild-type characteristics in vitro, as measured by cytopathic effect, plaque morphology, virus titres and growth kinetics. However, one mutant (FAdV-9D4), which lacked these ORFs and retained the packaging motifs, did not replicate at wild-type levels in vivo, as judged in infected eggs by virus titres in allantoic fluid and in infected chickens by antibody responses, virus titres in faeces and virus genome copy numbers in tissues. These findings indicate that some of the ORFs in this region, although dispensable in vitro, are important for in vivo replication of FAdV-9. INTRODUCTIONFowl adenoviruses (FAdVs) have a worldwide distribution, and some are important poultry pathogens that cause inclusion body hepatitis and hydropericardium syndrome in chickens (Adair & Fitzgerald, 2008). FAdV serotypes are grouped into five species (Fowl adenovirus A-E), all belonging to the genus Aviadenovirus, family Adenoviridae (Benkő et al., 2005). Complete nucleotide sequences are available for the genomes of FAdV-1 (CELO virus) and FAdV-9 (strain A-2A) (Chiocca et al., 1996;Ojkic & Nagy, 2000), and partial nucleotide sequences are also available for the left and right ends of the genomes of FAdV-2, -4, -8 and -10 (strain C-2B) (Corredor et al., 2006(Corredor et al., , 2008. None of the early genes at the left and right ends of the FAdV genomes are homologues of the E1, E3 and E4 genes of mastadenoviruses, which infect mammals Chiocca et al., 1996). In this study, the importance of ORFs at the left end of the FAdV-9 genome in virus replication in vitro and in vivo was investigated by analysing deletions, as a first step towards elucidating the roles of the early genes in this region. RESULTS Generation of deletionsThe ORF arrangement within the left-end genomic region of FAdV-9 and the features of all deletions are depicted in Fig. 1(a, b), respectively. Nine deletions were generated in the region within nt 250-4200 of the FAdV-9 genome as shown: FAdV-9D1 (D1194-2342), FAdV-9D2 (D854-2782), FAdV-9D3 (D854-2817), FAdV-9D4 (D491-2782), FAdV-9D5 (D491-2980), FAdV-9D6 (D491-4200), FAdV-9D7 (D400-2782), FAdV-9D8 (D330-2782) and FAdV-9D9 (D250-2782). Viable viruses were recovered following transfection of CH-SAH (chicken hepatoma) cells without complementation with only the FAdV-9D1, -9D2, -9D4 and -9D7 genomes at 5-7 days post-transfection (p.t.). The first viable deletion mutant, FAdV-9D1, was generated by Supplementary methods and a supplementary figure and table are available with the online version of this paper.

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“…Previously, we have shown that this cell line can support full FAdV-8 replication (Alexander et al, 1998). Eighty percent confluent CH-SAH cell monolayers in 60 mm Petri dishes were infected with FAdV-8 at an m.o.i.…”

Section: Rt-pcrmentioning

confidence: 99%