Enhancement of Enteric Adenovirus Cultivation by Viral Transactivator Proteins

Abstract

Human enteric adenoviruses (HAdVs; serotypes 40 and 41) are important waterborne and food-borne pathogens. However, HAdVs are fastidious, are difficult to cultivate, and do not produce a clear cytopathic effect during cell culture within a reasonable time. Thus, we examined whether the viral transactivator proteins cytomegalovirus (CMV) IE1 and hepatitis B virus (HBV) X promoted the multiplication of HAdVs. Additionally, we constructed a new 293 cell line expressing CMV IE1 protein for cultivation assays. We analyzed the nucleic acid sequences of the promoter regions of both E1A and hexon genes, which are considered to be the most important regions for HAdV replication. Expression of either HBV X or CMV IE1 protein significantly increased the promoter activities of E1A and hexon genes of HAdVs by as much as 14-fold during cell cultivation. The promotion of HAdV expression was confirmed by increased levels of both adenoviral DNA and mRNA expression. Finally, the newly developed 293 cell line expressing CMV IE1 protein showed an increase in viral DNA ranging from 574% to 619% compared with the conventional 293 cell line. These results suggest that the newly constructed cell line could be useful for efficient cultivation and research of fastidious HAdVs.

Molecular characterization and multiple infections of rotavirus, norovirus, sapovirus, astrovirus and adenovirus in outpatients with sporadic gastroenteritis in Shanghai, China, 2010–2011

“…These data suggest that different HAdV genotypes could trigger similar symptoms in children; however, further research is required. Consistent with studies in Lanzhou and Tianjin in China, Japan and Ghana [9,12,62,67,68], we found that other types of HAdV were detected less frequently in children presenting with diarrhea, including HAdV-12, HAdV-2, HAdV-5 and HAdV-19. This phenomenon indicates that other ''nonenterovirus'' pathogens are significant in children with diarrhea, which requires additional attention from physicians.…”

Construction of an infectious clone of human adenovirus type 41

“…As mentioned in the introduction section, HAdV-41 is an interesting type of human adenoviruses that deserves further study. Recently, several cell lines have been developed to amplify HAdV-F, including 293-SV5/V (293 cells transduced with the V protein gene of the paramyxovirus simian virus 5) [20], 293-CMV (constitutively expressing cytomegalovirus IE1 protein) [12], 293-ORF6, and 2V6.11 (inducibly expressing the HAdV-5 E4orf6 gene) [15,25]. Some of these cell lines were established to isolate viruses from patients or the environment.…”

Enhancement of enteric adenovirus cultivation in a novel Ras-overexpressing cell line

“…Our work with the cytomegalovirus (CMV) transactivator protein IE1 improved AdV cultivation by promoting the expression of the essential viral genes, E1A and hexon (Kim et al, 2010). Transient transfection of 293 cells with CMV IE1 showed two and four times higher levels of viral DNA and RNA, respectively, compared with the 293 control cells.…”

“…The Ras protein level in the new cells was confirmed by Western blotting as described previously (Kim et al, 2010). Antibodies used were anti-Ras mAb (1 : 2000 dilution; Millipore), anti-b-actin antibody (1 : 10 000 dilution; Abcam) and secondary anti-mouse-HRP antibody (1 : 5000 dilution; Abcam).…”

“…After a 5 min post-pulse incubation on ice, the cell suspension was grown in T25 flasks under the same culture conditions as above. Transformed cells were selected under G418 (Gibco) as described previously (Kim et al, 2010). Introduction and expression of the H-Ras gene were confirmed by PCR and RT-PCR.…”