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Journal of Virological Methods
journal homepage: www.elsevier.com/locate/jviromet
Propagation of avian metapneumovirus subtypes A and B using chicken embryo related and other cell systems
Lia Treptow Coswig a , Márcia Bianchi dos Santos b,∗ , Hafez Mohamed Hafez c , Helena Lage Ferreira d , Clarice WeisArns b
Ministry of Agriculture, Livestock and Food Supply, Av. General San Martin, 1000 Recife, PE 50630-060, Brazil Laboratory of Virology, Institute of Biology, State University of Campinas – UNICAMP, Campinas, SP, Brazil Institute of Poultry Diseases, Faculty of Veterinary Medicine of the “Freie Universität Berlin”, Königsweg 63, Berlin 14163, Germany d Avian Virology and Immunology,CODA-CERVA-VAR, Groeselenberg 99, B-1180 Uccle, Brussels, Belgium
a b s t r a c t
Article history: Received 20 August 2009 Received in revised form 15 February 2010 Accepted 18 February 2010 Available online 26 February 2010 Keywords: Avian metapneumovirus Cell propagation CER
Primary isolation of avian metapneumovirus (aMPV) is carried out using tracheal organ culture (TOC) or chicken embryonatedeggs with subsequent adaptation in chicken embryo ﬁbroblasts (CEF) or Vero cultures. This study was conducted to evaluate six different cell lines and two avian culture systems for the propagation of aMPV subtypes A and B. The chicken embryo related (CER) cells were used successfully for primary isolation. In addition to Vero and baby hamster kidney (BHK-21) cells, CER cells were also shown to be themost appropriate for propagation of aMPV considering high titres. Propagation of A and B subtypes in CEF and TOC remained efﬁcient after the primary isolation and several passages of viruses in the CER cell line. The growth curves were created using CER, Vero and BHK-21 cell lines. Compared with growth, both yielded higher titres in CER cells during the ﬁrst 30 h after infection, but nosigniﬁcant difference was observed in the results obtained from CER and Vero cells. This data show that CER cells are adequate for aMPV subtypes A and B propagation, giving similar results to Vero cells. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Avian metapneumovirus (aMPV) is classiﬁed as a member of the Metapneumovirus genus within the paramyxoviridae family of viruses (Pringle, 1998). Thevirus causes upper respiratory tract infection in turkeys and chickens of all ages and is present in a wide range of countries (Jones, 1996; Cook, 2000). aMPV has been classiﬁed into four subtypes A, B, C and D based on antigenic and genetic characterization (Toquin et al., 2000; Dar et al., 2001; Alvarez et al., 2003). The virus was ﬁrst described in South Africa in 1978 (Buys and Du Preez,1980) and was reported thereafter in Europe (Hafez et al., 2000), the United States (Cook et al., 1999), the Middle East, Asia and Africa (Njenga and Seal, 2003; Owoade et al., 2008), Brazil (Arns and Hafez, 1992; D’Arce et al., 2005) and other parts of the world (Buys et al., 1989a,b; Cook, 2002). aMPV infections can be detected by serology and molecular methods; however
∗ Corresponding author at:Laboratório de Virologia, Departamento de Microbiologia e Imunologia, Instituto de Biologia, Universidade Estadual de Campinas (UNICAMP), P.O. Box 6109, CEP 13081-970, Campinas, SP, Brazil. Tel.: +55 19 3521 6258; fax: +55 19 3521 6276. E-mail addresses: firstname.lastname@example.org (L.T. Coswig), email@example.com (M.B.d. Santos), firstname.lastname@example.org (H.M. Hafez),Helena.email@example.com (H.L. Ferreira), firstname.lastname@example.org (C.W. Arns). 0166-0934/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.jviromet.2010.02.018
they do not provide a sample of live virus. If such a sample is required, virus isolation is to be performed (Goyal et al., 2000; Shin et al., 2000). The methods of choice for primary isolation of aMPV are either the use of...