Isolation of influenza viruses in MDCK 33016PF cells and clearance of contaminating respiratory viruses

• Published in: Vaccine Vol. 30; no. 3; pp. 517 - 522
• Main Authors: Roth, Bernhard, Mohr, Hannah, Enders, Martin, Garten, Wolfgang, Gregersen, Jens-Peter
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier Ltd 11.01.2012; Elsevier Limited
• Subjects: Adenovirus; Adenoviruses; Adventitious viruses; Allergy and Immunology; Animals; Binding sites; Blood; Bocaparvovirus; Bocavirus; Cell culture; Cell Line; Coinfection - virology; Coronavirinae; Coronavirus; Dogs; Eggs; Enterovirus; Germany; Humans; Influenza; Influenza vaccine; Influenza virus; inoculum; MDCK cells; Multiplex Polymerase Chain Reaction; Mutation; Orthocoronavirinae; Orthomyxoviridae; Parainfluenza virus; polymerase chain reaction; Respiratory syncytial virus; Respiratory tract; Respiratory Tract Infections - diagnosis; Respiratory Tract Infections - virology; Rhinovirus; screening; Vaccines; Virology - methods; Virus Cultivation - methods; Virus Diseases - diagnosis; Virus Diseases - virology; Virus isolation; Viruses; Viruses - genetics; Viruses - growth & development; Viruses - isolation & purification; Germany; MDCK cells; Influenza vaccine; Virus isolation; Adventitious viruses
• ISSN: 0264-410X; 1873-2518; 1873-2518
• DOI: 10.1016/j.vaccine.2011.11.063

► Multiplex PCR screening of clinical specimens from acute respiratory tract infections. ► Viruses found were influenza, entero/rhino, corona, adeno, boca, PI, RSV, and hMPV. ► After 3 or less passages in MDCK 33016 PF cells, only influenza virus was found. ► Co-infections together with influenza virus were lost within 2–3 MDCK passages. ► MDCK cells represent a barrier for co-infecting viruses in influenza vaccine strains. This paper summarizes results obtained by multiplex PCR screening of human clinical samples for respiratory viruses and corresponding data obtained after passaging of virus-positive samples in MDCK 33016PF cells. Using the ResPlexII v2.0 (Qiagen) multiplex PCR, 393 positive results were obtained in 468 clinical samples collected during an influenza season in Germany. The overall distribution of positive results was influenza A 42.0%, influenza B 38.7%, adenovirus 1.5%, bocavirus 0.5%, coronavirus 3.3%, enterovirus 5.6%, metapneumovirus 1.0%, parainfluenza virus 0.8%, rhinovirus 4.1%, and respiratory syncytial virus (RSV) 2.5%. Double infections of influenza virus together with another virus were found for adenovirus B and E, bocavirus, coronavirus, enterovirus and for rhinovirus. These other viruses were rapidly lost upon passages in MDCK 33016PF cells and under conditions as applied to influenza virus passaging. Clinical samples, in which no influenza virus but other viruses were found, were also subject to passages in MDCK 33016PF cells. Using lower inoculum dilutions than those normally applied for preparations containing influenza virus (total dilution of the original sample of ∼104), the positive results for the different viruses turned negative already after 2 or 3 passages in MDCK 33016PF cells. These results demonstrate that, under practical conditions as applied to grow influenza viruses, contaminating viruses can be effectively removed by passages in MDCK cells. In combination with their superior isolation efficiency, MDCK cells appear highly suitable to be used as an alternative to embryonated eggs to isolate and propagate influenza vaccine candidate viruses.