Semi-quantitative RT-PCR-based assay, improved by Southern hybridization technique, for polarity-specific influenza virus RNAs in cultured cells.
Complementary (c) DNAs against viral (v) RNA of negative polarity and complementary and/or messenger (c/m) RNA of positive polarity for influenza virus hemagglutinin (HA) were synthesized from total cellular RNA extracted from influenza virus- and mock-infected cells using polarity-specific primers, respectively. HA vRNA and c/mRNA were amplified readily by polymerase chain reaction (PCR) from influenza virus-infected cells during a virus productive period; however, non-specific PCR product was prone to amplification from mock-infected cells and cells at once after virus infection. Southern blots of the PCR products were hybridized with biotinylated DNA probe, which enabled the generation of specific signals to HA vRNA and c/mRNA. Mock-infected cells produced no signals. Furthermore, titration analyses revealed linear relationships between amount of target RNAs and generated signals. Accordingly, Southern hybridization made possible the quantitation of specific PCR products for HA vRNA and c/mRNA in cell culture and proved the lack of HA RNAs in mock-infected cells in the absence of virus. The RT-PCR based assay combined with Southern hybridization methodology was useful with respect for investigating the processes of replication and transcription of viral genes in cell culture before and during the virus productive period.
D000818:Animals; D000998:Antiviral Agents / Q000494:pharmacology; D015139:Blotting, Southern / Q000379:methods*; D016764:Cell Polarity; D002478:Cells, Cultured; D002642:Chick Embryo; D019267:Hemagglutinin Glycoproteins, Influenza Virus / Q000235:genetics / Q000378:metabolism; D006801:Humans; D009980:Influenza A virus / Q000187:drug effects / Q000302:isolation & purification* / Q000502:physiology; D008826:Microbial Sensitivity Tests; D012367:RNA, Viral / Q000032:analysis*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012254:Ribavirin / Q000494:pharmacology
D000998:Antiviral Agents; D019267:Hemagglutinin Glycoproteins, Influenza Virus; D012367:RNA, Viral; D012254:Ribavirin