Comparison of Methods for Detection of Vaccinia Virus in Patient Specimens

Fedorko, Daniel P.; Preuss, Jeanne C.; Fahle, Gary A.; Li, Li; Fischer, Steven H.; Hohman, Patricia; Cohen, Jeffrey I.

doi:10.1128/jcm.43.9.4602-4606.2005

Cited by 14 publications

(5 citation statements)

References 24 publications

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“…Vaccinia virus WR strain [ 28 ] was used to infect HeLa and HuH 7 cells in this study, following previously published procedures for virus amplification and plaque assay [ 22 , 29 ]. Cytosine arabosinide (ara C), where used, was added to the cells at a concentration of 40 μg/ml.…”

Section: Methodsmentioning

confidence: 99%

Cleavage of Dicer Protein by I7 Protease during Vaccinia Virus Infection

Chen

Lin

et al. 2015

PLoS ONE

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Dicer is the key component in the miRNA pathway. Degradation of Dicer protein is facilitated during vaccinia virus (VV) infection. A C-terminal cleaved product of Dicer protein was detected in the presence of MG132 during VV infection. Thus, it is possible that Dicer protein is cleaved by a viral protease followed by proteasome degradation of the cleaved product. There is a potential I7 protease cleavage site in the C-terminus of Dicer protein. Indeed, reduction of Dicer protein was detected when Dicer was co-expressed with I7 protease but not with an I7 protease mutant protein lack of the protease activity. Mutation of the potential I7 cleavage site in the C-terminus of Dicer protein resisted its degradation during VV infection. Furthermore, Dicer protein was reduced dramatically by recombinant VV vI7Li after the induction of I7 protease. If VV could facilitate the degradation of Dicer protein, the process of miRNA should be affected by VV infection. Indeed, accumulation of precursor miR122 was detected after VV infection or I7 protease expression. Reduction of miR122 would result in the suppression of HCV sub-genomic RNA replication, and, in turn, the amount of viral proteins. As expected, significant reduction of HCVNS5A protein was detected after VV infection and I7 protease expression. Therefore, our results suggest that VV could cleave Dicer protein through I7 protease to facilitate Dicer degradation, and in turn, suppress the processing of miRNAs. Effect of Dicer protein on VV replication was also studied. Exogenous expression of Dicer protein suppresses VV replication slightly while knockdown of Dicer protein does not affect VV replication significantly.

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Section: Methodsmentioning

confidence: 99%

Cleavage of Dicer Protein by I7 Protease during Vaccinia Virus Infection

Chen

Lin

et al. 2015

PLoS ONE

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“…Vaccinia virus WR strain was used to infect HeLa cells in this study, following previously published procedures for virus amplification and plaque assay [ 23 , 24 ]. Cytosine arabosinide (ara C), where used, was added to the cells at a concentration of 40 μg/ml [ 25 ].…”

Section: Methodsmentioning

confidence: 99%

Increased ATP generation in the host cell is required for efficient vaccinia virus production

Chang¹,

Li²,

Hsu³

et al. 2009

J Biomed Sci

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To search for cellular genes up-regulated by vaccinia virus (VV) infection, differential displayreverse transcription-polymerase chain reaction (ddRT-PCR) assays were used to examine the expression of mRNAs from mock-infected and VV-infected HeLa cells. Two mitochondrial genes for proteins that are part of the electron transport chain that generates ATP, ND4 and CO II, were up-regulated after VV infection. Up-regulation of ND4 level by VV infection was confirmed by Western blotting analysis. Up-regulation of ND4 was reduced by the MAPK inhibitor, apigenin, which has been demonstrated elsewhere to inhibit VV replication. The induction of ND4 expression occurred after viral DNA replication since ara C, an inhibitor of poxviral DNA replication, could block this induction. ATP production was increased in the host cells after VV infection. Moreover, 4.5 μM oligomycin, an inhibitor of ATP production, reduced the ATP level 13 hr after virus infection to that of mock-infected cells and inhibited viral protein expression and virus production, suggesting that increased ATP production is required for efficient VV production. Our results further suggest that induction of ND4 expression is through a Bcl-2 independent pathway.

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“…This method is laborious due to required additional specimen manipulation, including fixing, staining and labeling [reviewed by Trindade et al, 2007]. Molecular and immunological techniques, including real-time PCR [Putkuri et al, 2009], nested and semi-nested PCR [Sá nchez-Seco et al, 2006;Damaso et al, 2007], PCR-RFLP [Meyer et al, 1997], and Western blot assays [Fedorko et al, 2005] have been applied widely in OPV research. The use of real-time PCR for the detection of VACV directly from lesions without DNA or virus manipulation was described by de Souza Trindade et al [2008].…”

Section: Introductionmentioning

confidence: 99%

Rapid detection of Orthopoxvirus by semi‐nested PCR directly from clinical specimens: A useful alternative for routine laboratories

Abrahão

Drumond

Trindade

et al. 2010

Journal of Medical Virology

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Orthopoxvirus (OPV) has been associated with worldwide exanthematic outbreaks, which have resulted in serious economic losses as well as impact on public health. Although the current classical and molecular methods are useful for the diagnosis of OPV, they are largely inaccessible to unsophisticated clinical laboratories. The major reason for the inaccessibility is that they require both virus isolation and DNA manipulation. In this report, a rapid, sensitive and low-cost semi-nested PCR method is described for the detection of OPV DNA directly from clinical specimens. A set of primers was designed to amplify the conserved OPV vgf gene. The most useful thermal and chemical conditions were selected and minimum non-inhibitory dilutions were determined. More than 100 Brazilian Vaccinia virus (VACV) field clinical specimens were tested using this semi-nested PCR in order to confirm its applicability. Cowpox virus was also detected by PCR from the ear scabs of scarified Balb/c mice. In addition, the method was highly sensitive for the detection of VACV DNA in murine blood and excreta, which are among the suggested reservoirs of OPV. Together, these data suggest that semi-nested PCR can be used for initial screening for OPV and as a routine diagnostic laboratory method.

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Comparison of Methods for Detection of Vaccinia Virus in Patient Specimens

Cited by 14 publications

References 24 publications

Cleavage of Dicer Protein by I7 Protease during Vaccinia Virus Infection

Cleavage of Dicer Protein by I7 Protease during Vaccinia Virus Infection

Increased ATP generation in the host cell is required for efficient vaccinia virus production

Rapid detection of Orthopoxvirus by semi‐nested PCR directly from clinical specimens: A useful alternative for routine laboratories

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