Detection of Infectious Adenovirus in Cell Culture bymRNA ReverseTranscription-PCR

Ko, GwangPyo; Cromeans, Theresa L.; Sobsey, Mark D.

doi:10.1128/aem.69.12.7377-7384.2003

Cited by 93 publications

(93 citation statements)

References 31 publications

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“…The rapid culture centrifugation method shows only a slight difference (1%) from conventional culture, and additional serotyping or a genus-specific enzyme-linked immunosorbent assay (ELISA) is still required. The mRNA RT-PCR method was targeted on mRNA as an index of infectious HAdVs (17,18). These previous studies were able to rapidly detect infectious HAdV by targeting viral mRNA that was produced by infectious virus during multiplication.…”

Section: Discussionmentioning

confidence: 99%

Enhancement of Enteric Adenovirus Cultivation by Viral Transactivator Proteins

Kim

Lim

2010

Appl Environ Microbiol

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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.

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Section: Discussionmentioning

confidence: 99%

Enhancement of Enteric Adenovirus Cultivation by Viral Transactivator Proteins

Kim

Lim

2010

Appl Environ Microbiol

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“…No sensitive cell lines are available; alternatively, the presence of the opposite strand of these singlestrand viruses could be determined to establish if replication took place. This has been described for adenoviruses but yet has to be developed for noroviruses (26). In absence of such methods, we estimated the virus concentrations by RT-PCR on 10-fold serial dilutions of the extracted RNA as a semiquantitative approach.…”

Section: Discussionmentioning

confidence: 99%

Presence of Noroviruses and Other Enteric Viruses in Sewage and Surface Waters in The Netherlands

Lodder

Husman

2005

Appl Environ Microbiol

341

201

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Since virus concentrations in drinking waters are generally below the detection limit, the infectious risk from drinking water consumption requires assessment from the virus concentrations in source waters and removal efficiency of treatment processes. In this study, we estimated from reverse transcription-PCR on 10-fold serially diluted RNA that noroviruses, the most prevalent waterborne gastroenteritis agents, were present at 4 (0.2 to 38) to 4,900 (303 to 4.6 ؋ 10 4 ) PCR-detectable units (PDU) per liter of river water (ranges are given in parentheses). These virus concentrations are still high compared with 896 to 7,499 PDU/liter of treated sewage and 5,111 to 850,000 PDU/liter in raw sewage. Sequencing analyses designated human norovirus GGII.4 Lordsdale as the most prevalent strain in the sampling period 1998 to 1999 in both sewage and surface waters. Other GGII strains were also very abundant, indicating that the majority of the virus contamination was derived from urban sewage, although very divergent strains and one animal strain were also detected in the surface and sewage waters. Rotaviruses were also detected in two large rivers (the Maas and the Waal) at 57 to 5,386 PDU/liter. The high virus concentrations determined by PCR may in part be explained by the detection of virus RNA instead of infectious particles. Indeed, reoviruses and enteroviruses that can be cultured were present at much lower levels, of 0.3 to 1 and 2 to 10 PFU/liter, respectively. Assuming 1% of the noroviruses and rotaviruses to be infectious, a much higher disease burden than for other viruses can be expected, not only because of the higher levels but also because of these viruses' higher infectivity and attack rates.

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“…Direct antigen detection by immunofluorescence techniques or enzyme immunoassays has been reported but is often too insensitive for detecting low concentrations of viruses (17). PCR assays based on amplification of either viral DNA or RNA provide improved sensitivity and specificity over immunoassay, but these techniques unfortunately only indicate the presence or absence of viruses in a sample and do not provide any information on infectivity, which is directly related to health risk (6). This is a critical requirement, as many inactivated viruses that carry the required antigens or genomic sequence could persist over a relatively long period in the environment (13).…”

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“…Although integrated cell culture-reverse transcription-PCR (RT-PCR) assay has been used to provide rapid detection of infectious viruses (6) by probing the presence of viral mRNA, this method requires additional mRNA extraction, RT-PCRs, and gel analysis, leading to the potential for contamination. Since a large portion of viral mRNA is synthesized early in the infectious cycle (1), in situ detection of viral mRNA directly from cell cultures may be used as a very sensitive and specific indicator for infectious viruses without amplification.…”

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confidence: 99%