Pseudoplusia includens single nucleopolyhedrovirus: Genetic diversity, phylogeny and hypervariability of the pif-2 gene Saluana R. Craveiro a,b , Fernando L. Melo a , Zilda Maria A. Ribeiro b , Bergmann M. Ribeiro a , Sônia Nair Báo a , Peter W. Inglis b,1 , Maria Elita B. Castro b,⇑,1 a Departamento de Biologia Celular, Universidade de Brasília-UnB, Brasília, DF, Brazil b Embrapa Recursos Genéticos e Biotecnologia, Parque Estação Biológica, W5 Norte Final, 70770-917 Brasília, DF, Brazil article info Article history: Received 8 April 2013 Accepted 22 August 2013 Available online 5 September 2013 Keywords: Pseudoplusia includens SNPV Genotypic variants pif-2 Gene polymorphisms Phylogeny abstract The soybean looper (Pseudoplusia includens Walker, 1857) has become a major pest of soybean crops in Brazil. In order to determine the genetic diversity and phylogeny of variants of Pseudoplusia includens sin- gle nucleopolyhedrovirus (PsinSNPV-IA to -IG), partial sequences of the genes lef-8, lef-9, pif-2, phr and polh were obtained following degenerate PCR and phylogenetic trees constructed using maximum parsimony and Bayesian methods. The aligned sequences showed polymorphisms among the isolates, where the pif- 2 gene was by far the most variable and is predicted to be under positive selection. Furthermore, some of the pif-2 DNA sequence mutations are predicted to result in significant amino acid substitutions, possibly leading to changes in oral infectivity of this baculovirus. Cladistic analysis revealed two closely related monophyletic groups, one containing PsinNPV isolates IB, IC and ID and another containing isolates IA, IE, IF and IG. The phylogeny of PsinSNPV in relation to 56 other baculoviruses was also determined from the concatenated partial LEF-8, LEF-9, PIF-2 and POLH/GRAN deduced amino acid sequences, using max- imum-parsimony and Bayesian methods. This analysis clearly places PsinSNPV with the Group II Alpha- baculovirus, where PsinSNPV is most closely related to Chrysodeixis chalcites NPV and Trichoplusia ni SNPV. Ó 2013 Elsevier Inc. All rights reserved. 1. Introduction Pseudoplusia includens single nucleopolyhedrovirus (PsinSNPV) is a baculovirus pathogenic to the soybean looper, Pseudoplusia includens (Lepidoptera: Noctuidae) (Walker, 1857) (synonym: Chrysodeixis includens). The P. includens caterpillar is an emerging pest of soybeans, and also of various other crops of economic importance in Brazil, such as cotton, beans, potatoes, tomatoes, to- bacco, sunflower and other vegetables (Bueno et al., 2009). Seven PsinSNPV isolates (IA to IG) were obtained from P. includens larvae collected in soybean and cotton crops in Brazil and Guatemala. Using restriction endonuclease analysis (REN) and virulence bioas- say, these seven isolates showed different restriction patterns with the presence of sub-molar bands and possessed varying pathoge- nicity, where isolates IA, IE and IF were more virulent than others isolates (Alexandre et al., 2010). Baculoviruses are a large group of arthropod-specific DNA viruses that infect mostly the larval stages of insects in the orders Lepidoptera, Hymenoptera and Diptera (Herniou et al., 2012; Jehle et al., 2006a). Occlusion bodies (OBs) are viral particles that permit virus survival in the environment. OBs are composed of a crystal- line matrix of protein (polyhedrin in nucleopolyhedroviruses – NPVs and granulin in granuloviruses – GVs) that occludes the enveloped virions (Adams and McClintock, 1991; Bilimoria, 1991; Boucias and Pendland, 1998). The nucleopolyhedroviruses exhibit OBs called polyhedra, composed of several virions, which may con- tain single (SNPV) or multiple (MNPV) nucleocapsids per envelope. The granuloviruses (GVs) exhibit OBs in the form of granules, con- taining one or, uncommonly, two or three virions per granule (Frie- sen and Miller, 2001; Herniou et al., 2012). The Baculoviridae family is divided into four genera: Alphabaculovirus – lepidopteran-spe- cific NPVs, subdivided into Group I and Group II based on the type of budded virus (BV) fusogenic protein; Betabaculovirus – lepidop- teran-specific GVs; Gammabaculovirus – hymenopteran-specific NPVs; and Deltabaculovirus – dipteran-specific NPVs (Carstens and Ball, 2009; Herniou et al., 2012; Jehle et al., 2006a). Baculoviruses are widely and successfully used as biocontrol agents of insect pests of agricultural, forest and vegetable produc- tion systems (Copping and Menn, 2000; Erlandson, 2008; Moscardi et al., 2011; Souza et al., 2007; Szewczyk et al., 2006, 2009, 2011). High virulence, specific host range, stability and low environmen- tal impact are considered to be advantages of using baculovirus insecticides. These viruses are also used as biotechnological tools for the production of recombinant proteins in insect cells and for 0022-2011/$ - see front matter Ó 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jip.2013.08.005 ⇑ Corresponding author. Fax: +55 61 3448 4673. E-mail address: elita.castro@embrapa.br (M.E. B. Castro). 1 These authors contributed equally to this work. Journal of Invertebrate Pathology 114 (2013) 258–267 Contents lists available at ScienceDirect Journal of Invertebrate Pathology journal homepage: www.elsevier.com/locate/jip