Improved In Vitro Cultivation of Swine Influenza Virus

Diagnosis of swine influenza virus (SIV) involves virus isolation in embryonated chicken eggs (ECE) and/or Madin-Darby canine kidney (MDCK) cells with trypsin added to the maintenance medium. Both trypsin and diethylaminoethyl (DEAE) dextran have been reported to enhance the attachment, entry, and replication of a number of viruses. Similarly, centrifugation of virus inoculum in shell vials has been found to be useful in growing certain viruses. In this study, we evaluated 8 different cell types for the propagation of SIV with or without the addition of trypsin or DEAE-dextran. In addition, shell vial centrifugation was evaluated to increase virus titers. The cell cultures used were MDCK, porcine turbinate (PT), swine testis (ST), St. Jude porcine lung epithelial cells (SJPL), porcine kidney (PK), feline kidney (CRFK), Vero, and horse stem cells (H-1). None of the cell cultures supported the growth of SIV with or without added trypsin, except for MDCK cells with 0.15% trypsin. The addition of DEAE-dextran did not improve the growth of SIV in any cell type. Similarly, shell vial centrifugation did Intern J Appl Res Vet Med • Vol. 3, No. 2, 2005 124 pigs followed by hemagglutination-inhibition (HI) and neuraminidase (NA) assays for subtyping. The standard method for isolation of SIV is inoculation of 10to 11-day old embryonated chicken eggs (ECE). B e c a u s e of the cumbersome nature of this method, SIV isolation is now done in Madin-Darby canine kidney (MDCK) cells using trypsin as an additive. , 6 It has been known since 1975 that the addition of trypsin to culture media stimulates the growth of influenza A v i r u s e s . Recently Kessler et al. r e p o r t e d the growth of influenza A and B viruses in the absence of trypsin in serum-free media MDCK cells. Clavijo et al. r e c o m m e n d e d the use of both ECE and MDCK cells for the growth of SIV in media containing trypsin. Other studies in the past have indicated that the addition of polycations, for example, diethylaminoethyl (DEAE)-dextran, and the use of shell vial centrifugation may also increase the yield of many v i r u s e s . 0 – 1 5 We are not familiar with any study in which the growth of SIV has been evaluated in various cell lines, in the presence or absence of trypsin or DEAE-dextran, and using shell vial centrifugation. This study was therefore undertaken to fulfill this need. MATERIALS AND METHODS