Please cite this article in press as: Jakubowska, A., et al., Enhancing the multiplication of nucleopolyhedrovirus in vitro by manipulation of the pH. J. Virol. Methods (2009), doi:10.1016/j.jviromet.2009.06.020 ARTICLE IN PRESS G Model VIRMET-10935; No. of Pages 5 Journal of Virological Methods xxx (2009) xxx–xxx Contents lists available at ScienceDirect Journal of Virological Methods journal homepage: www.elsevier.com/locate/jviromet Enhancing the multiplication of nucleopolyhedrovirus in vitro by manipulation of the pH Agata Jakubowska a,b , Juan Ferré a , Salvador Herrero a,∗ a Department of Genetics, Universitat de València, 46100 Burjassot (Valencia), Spain b Department of Biocontrol and Quarantine, Institute of Plant Protection, 60318 Poznan, Poland Article history: Received 14 April 2009 Received in revised form 17 June 2009 Accepted 23 June 2009 Available online xxx Keywords: Baculovirus Insect virus Cell culture Virus multiplication Endocytosis abstract Insect nucleopolyhedroviruses (NPVs) are studied widely as agents for biological control, as expression vectors for the production of heterologous proteins, and as transduction vectors for gene therapy applica- tions. Most of these applications rely on the existence of cell lines that allow in vitro multiplication of the virus. The influence of pH in the medium culture on the multiplication of SeMNPV, HearSNPV and AcM- NPV in different cell culture lines was investigated. The study showed a strong influence of the medium pH on the virus multiplication with the best results at pH 6.5, about half pH unit above the pH of insect culture media used most commonly. Additional experiments using a recombinant AcMNPV, expressing the green fluorescent protein, suggested that the enhanced virus multiplication at pH 6.5 is due mainly to a facilitated entry of the budded virions into the cells. © 2009 Elsevier B.V. All rights reserved. 1. Introduction Baculoviruses are large, double-stranded DNA viruses infect- ing invertebrates, mainly insects from the order Lepidoptera, but also from the Diptera and Hymenoptera orders. According to the last ICTV report (Mayo et al., 2005), the family Baculoviri- dae is classified into two genera, Nucleopolyhedrovirus (NPV) and Granulovirus (GV). However, Jehle et al. (2006) proposed a re- classification and re-naming of the baculovirus genera based on phylogenetic evidence and genome composition, and on morpho- logical and pathological data. This new classification contains: alphabaculoviruses (including lepidopteran-specific nucleopoly- hedroviruses), betabaculoviruses (including lepidopteran-specific granuloviruses), gammabaculoviruses (comprising hymenopteran- specific nucleopolyhedroviruses), and deltabaculoviruses (includ- ing dipteran-specific nucleopolyhedroviruses). Baculoviruses are studied widely as agents for biological control, as expression vectors for heterologous protein production, and as transduction vectors for gene therapy. During the baculovirus infec- tion cycle in susceptible larvae, two types of virions are produced. One virion phenotype, contained in the viral occlusion body (OB) and known as the occlusion-derived virion (ODV), infects midgut ∗ Corresponding author at: Departament de Genètica, Universitat de València, Dr. Moliner 50, 46100 Burjassot (Valencia), Spain. Tel.: +34 96 354 3006; fax: +34 96 354 3029. E-mail address: salvador.herrero@uv.es (S. Herrero). epithelial cells and is responsible for the oral infectivity of the virus. A second phenotype is the budded virion (BV), which is responsi- ble for the cell-to-cell propagation of the infection throughout the larvae after the establishment of the infection in the midgut. As a consequence of their different roles in the infection cycle, the phe- notypes differ in their lipid and protein composition and in their mechanism of cell entry (Braunagel et al., 2003; Deng et al., 2007; Liu et al., 2008). ODVs enter into the cell by fusioning their mem- brane with the cellular plasma membrane. This fusion occurs in the insect midgut milieu and, possibly due to the adaptation to the particular alkaline environment of Lepidoptera’s midgut, the fusion occurs more efficiently under alkaline conditions (Horton and Burand, 1993). In contrast to ODVs, BVs enter the cell by endocy- tosis after attachment to the cell membrane. Evidence suggests that endocytosis is mediated by the presence of specific receptors (Wang et al., 1997; Zhou and Blissard, 2008), although some studies also suggest that electrostatic interaction could mediate endocytosis in the absence of specific receptors (Barsoum, 1999). On the basis of gene phylogenies the lepidopteran-specific NPVs have been divided into two groups, group I NPVs and group II NPVs (Zanotto et al., 1993; Bulach et al., 1999). This grouping also applies to the type of viral protein that mediates the entry of BVs. In group I NPV, such as Autographa californica multiple NPV (AcMNPV), the entrance is mediated by the envelope protein GP64 (Volkman, 1986). In group II NPV, such as Spodoptera exigua multiple NPV (SeMNPV) or Helicoverpa armigera single NPV (HearNPV), the entry is mediated by the F-protein (IJkel et al., 2000). Recombinant viruses lacking the expression of these proteins have lost their capacity for 0166-0934/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.jviromet.2009.06.020