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1.
Biologicals ; 59: 29-36, 2019 May.
Article in English | MEDLINE | ID: mdl-30992161

ABSTRACT

The utilization of the current combination of in vitro, in vivo and PCR assays for the identification of adventitious viruses in production cells has a limited range of detection. While Next Generation Sequencing (NGS) has a broader breadth of detection, it is unable to differentiate sequences from replicating viruses versus background inert sequences. In order to improve NGS specificity, we have designed a new NGS approach which targets subsets of viral RNAs only synthesized during cell infection. In order to evaluate the performance of this approach for detecting low levels of adventitious viruses, we selected two difficult virus/cell systems. This included B95-8 cells persistently infected by Human herpesvirus 4 (HHV-4) and serially diluted into HHV-4 negative Ramos cells and Madin-Darby bovine kidney cells with an early infection produced via a low dose of Bovine viral diarrhea virus. We demonstrated that the sensitivity of our RNA NGS approach was equivalent to targeted PCR with an increased specificity for the detection of viral infection. We were also able to identify a previously undetected Murine Leukemia Virus contaminant in Ramos cells. Based on these results, we conclude that this new RNA NGS approach is suitable for conducting viral safety evaluations of cells.


Subject(s)
High-Throughput Nucleotide Sequencing/methods , RNA, Viral/genetics , Sequence Analysis, RNA/methods , Viruses/genetics , Animals , Cattle , Cell Line , Cell Line, Tumor , Herpesvirus 4, Human/genetics , Herpesvirus 4, Human/physiology , Humans , Leukemia Virus, Murine/genetics , Leukemia Virus, Murine/physiology , Mice , Reproducibility of Results , Reverse Transcriptase Polymerase Chain Reaction , Sensitivity and Specificity , Virus Diseases/diagnosis , Virus Diseases/virology , Viruses/classification
2.
J Virol ; 76(23): 11920-30, 2002 Dec.
Article in English | MEDLINE | ID: mdl-12414934

ABSTRACT

The consequences of a hepatitis A virus (HAV) infection on cell-based antiviral responses and the interactions between virus and host cells resulting in persistent infections are poorly understood. In this report, we show that HAV does inhibit double-stranded (dsRNA)-induced beta interferon (IFN-beta) gene expression by influencing the IFN-beta enhanceosome, as well as dsRNA-induced apoptosis, which suggests that both effects may be connected by shared viral and/or cellular factors. This ability of HAV, which preserves the sites of virus production for a longer time, may allow the virus to establish an infection and may be the presupposition for setting up persistent infections. Our results suggest that the inhibitory effect of HAV on the cellular defense mechanisms might not be sufficient to completely prevent the antiviral reactions, which may be induced by accumulating viral dsRNA, at a later stage of infection. However, HAV seems to counteract this situation by downregulation of viral replication and in the following production of viral dsRNA. This ability of noncytopathogenic HAV acts dominantly on cytopathogenic HAV in trans. The downregulation might ensure the moderate replication which seems necessary for inhibition of the antiviral mechanisms by HAV and therefore for the persistent state of the HAV infection.


Subject(s)
Hepatitis A virus/immunology , Hepatitis A virus/pathogenicity , RNA, Double-Stranded/immunology , Animals , Apoptosis , Cell Line , Cytopathogenic Effect, Viral , Enhancer Elements, Genetic , Gene Expression , Hepatitis A/genetics , Hepatitis A/immunology , Hepatitis A/pathology , Hepatitis A/virology , Hepatitis A virus/genetics , Hepatitis A virus/physiology , Humans , Interferon-beta/biosynthesis , Interferon-beta/genetics , Macaca mulatta , RNA, Messenger/genetics , RNA, Messenger/metabolism , Virulence , Virus Replication
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